CN105973876B - It is a kind of detect dopamine electrochemical luminescence sensor and its preparation and application - Google Patents
It is a kind of detect dopamine electrochemical luminescence sensor and its preparation and application Download PDFInfo
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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
- 238000004020 luminiscence type Methods 0.000 title claims abstract description 54
- 229960003638 dopamine Drugs 0.000 title claims abstract description 50
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 238000001514 detection method Methods 0.000 claims abstract description 25
- 239000000758 substrate Substances 0.000 claims abstract description 23
- 239000002131 composite material Substances 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 20
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims abstract description 18
- 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 16
- 102000010909 Monoamine Oxidase Human genes 0.000 claims abstract description 15
- 108010062431 Monoamine oxidase Proteins 0.000 claims abstract description 15
- 102000004190 Enzymes Human genes 0.000 claims abstract description 4
- 108090000790 Enzymes Proteins 0.000 claims abstract description 4
- 239000004020 conductor Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 40
- 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 17
- 239000002953 phosphate buffered saline Substances 0.000 claims description 17
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 12
- 238000002484 cyclic voltammetry Methods 0.000 claims description 10
- 229910021607 Silver chloride Inorganic materials 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 7
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 claims description 7
- 229910052697 platinum Inorganic materials 0.000 claims description 6
- 239000011521 glass Substances 0.000 claims description 5
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- 125000004122 cyclic group Chemical group 0.000 claims description 4
- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical class Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 230000004044 response Effects 0.000 claims description 4
- 239000000337 buffer salt Substances 0.000 claims description 3
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- 235000010339 sodium tetraborate Nutrition 0.000 claims description 2
- 238000004832 voltammetry Methods 0.000 claims 1
- 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
- 230000008569 process Effects 0.000 abstract description 9
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- 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
- 229940116269 uric acid Drugs 0.000 abstract description 7
- 238000004458 analytical method Methods 0.000 abstract description 6
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- 238000002796 luminescence method Methods 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 description 18
- 239000010931 gold Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 5
- 229910052737 gold Inorganic materials 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 4
- 229910000397 disodium phosphate Inorganic materials 0.000 description 4
- 230000005518 electrochemistry Effects 0.000 description 4
- 229910000162 sodium phosphate Inorganic materials 0.000 description 4
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 3
- 230000005611 electricity Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 229930182817 methionine Natural products 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 108091023037 Aptamer Proteins 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- VYLQGYLYRQKMFU-UHFFFAOYSA-N Ochratoxin A Natural products CC1Cc2c(Cl)cc(CNC(Cc3ccccc3)C(=O)O)cc2C(=O)O1 VYLQGYLYRQKMFU-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 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
- 150000001412 amines Chemical class 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000000694 effects Effects 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
- 238000005259 measurement Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
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- 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 2
- 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 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 238000006862 quantum yield reaction Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- DZGWFCGJZKJUFP-UHFFFAOYSA-N tyramine Chemical compound NCCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-N 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
- LEOJISUPFSWNMA-UHFFFAOYSA-N ABEI Chemical compound O=C1NNC(=O)C=2C1=CC(N(CCCCN)CC)=CC=2 LEOJISUPFSWNMA-UHFFFAOYSA-N 0.000 description 1
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 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
- 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
- 239000002211 L-ascorbic acid Substances 0.000 description 1
- 235000000069 L-ascorbic acid Nutrition 0.000 description 1
- 208000027089 Parkinsonian disease Diseases 0.000 description 1
- 206010034010 Parkinsonism Diseases 0.000 description 1
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 229930003268 Vitamin C Natural products 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
- 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
- 150000003943 catecholamines Chemical class 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 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
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000007772 electrode material Substances 0.000 description 1
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- 230000002708 enhancing effect Effects 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 230000006870 function Effects 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
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229960004502 levodopa Drugs 0.000 description 1
- 238000000504 luminescence detection Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
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- 238000009938 salting Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
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- 229960003732 tyramine Drugs 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
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- 239000011787 zinc oxide Substances 0.000 description 1
- 229960001296 zinc oxide Drugs 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
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- 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 present invention provides a kind of electrochemical luminescence sensor for detecting dopamine, the electrochemical luminescence sensor includes electrode, the electrode includes substrate, composite layer and decorative layer, the substrate is conductive material, the composite layer is grown on the conductive substrates surface, it is polymerized by luminol and benzidine sulfate, the decorative layer is made of the tyramine oxidase for modifying in composite layer surface.The present invention also proposes the preparation and application of the electrochemical luminescence sensor.Biologically active enzyme is used for electrochemiluminescence analysis by the present invention, can effectively solve the problems, such as electrochemical luminescence method poor selectivity.The preparation of electrochemical sensor of the present invention and detection method are simple, high sensitivity, are not influenced by uric acid and ascorbic acid etc. in dopamine detection process, and anti-interference is good.
Description
Technical field
The invention belongs to detection fields, and in particular to a kind of electrochemical luminescence sensor and its preparation and application.
Background technique
Dopamine is a kind of naturally occurring catecholamine, is a kind of important nerve conduction mediator in the mammalian body,
It is most important to many basic physiological functions and higher brain function.Dopamine has very important adjustment effect to body, it
The neurodegenerative diseases such as concentration level in vivo and Parkinsonism are closely related.In view of the important physiology of dopamine
Function, its content in vivo of Accurate Determining become particularly important for the prophylactic treatment of disease.
The method of on-line determination dopamine concentration has very much, including chemiluminescence, electrochemical process, liquid chromatogram connexus
The methods of spectrum, surface plasma body resonant vibration and surface-enhanced Raman, such as patent CN201310479300.84 zinc-oxide nano
Piece ball/foamy graphite alkene composite material is detected as electrode material for dopamine.These means are one existing in testing
Problem is that their costs are higher, and measurement process takes a long time, and some tests need to pre-process sample and needed
Special test environment.
The analysis method that electrochemiluminescence analysis method is combined as chemiluminescence with electrochemistry, due to its luminescence-producing reaction
Area of space concentrate on region near electrode surface, have many advantages, such as that the controlling of luminescence-producing reaction is strong and high sensitivity, because
This by analysis worker extensive concern.There is electrochemiluminescence analysis equipment to build simply, detect signal background low noise,
The advantages such as highly sensitive and wider detection range can be used to detect glucose, and the macromolecules such as beta-lactamase and DNA are organic
Object is described such as a kind of preparation method of the electrochemical luminescence sensor of nanocomposite of patent CN201510482877.3
A kind of preparation method of the electrochemical luminescence sensor of nanocomposite, by composite material luminol-AuBSA-anti-CEA
It is deposited on the electrode surface of gold, obtains the electrochemical luminescence immunosensor that there is targeting to antigen CEA.Patent
A kind of method of electrochemical luminescence aptamer sensor detection ochratoxin A of CN201010271247.9, by nano Au particle
Naked gold electrode surface is modified, then modifies upper single stranded DNA in working electrode surface, then will be marked with different luminol (ABEI)
Hydrogen peroxide is added to electrode surface in aptamers modification, electroluminescence signal is detected, to detect ochratoxin A.Patent
CN201510594214.03 provides the Electrochemiluminescsensor sensor based on methionine gold nano cluster, with methionine
The gold nano cluster material of protection is illuminator, and over cure acid ion is coreagent, the gold nano group that methionine is protected
Cluster is modified on glass-carbon electrode, carries out electrochemical luminescence test, and realize the detection to dopamine.The sensor is to dopamine
The range of linearity of detection is 0.1~4 μm of ol/L and 4~25 μm of ol/L, and detection is limited to 0.032 μm of ol/L.
In the research of Electrochemiluminescince detection dopamine concentration, preparing for detection sensor is particularly important.Due to Shandong
Minot and Polyluminol have electro-chemical activity and efficient luminescent properties, in answering for electrochemical luminescence detection dopamine concentration
With especially extensively.But the quantum yield of luminol and Polyluminol is not high, detection sensitivity is lower.Simultaneously in living body environment
Ascorbic acid and uric acid and the dopamine of high concentration coexist, and since the oxidizing potential of three is much like, are easy the inspection to dopamine
Survey interferes.Therefore studying highly sensitive and highly selective electrochemical luminescence sensor will promote dopamine detection to lead significantly
The development in domain.
Summary of the invention
The present invention innovatively propose it is a kind of for dopamine detection electrochemical luminescence sensor, its object is to gram
Take that existing dopamine detection method involves great expense, time-consuming, sensitivity is low, the disadvantages of poor selectivity.In view of the above problems, this hair
It is bright to polymerize luminol and benzidine sulfate, the Photophysics of luminol are modulated, with benzidine sulfate to improve it in DOPA
Quantum yield in amine detection, enhances luminous intensity, to improve its detection sensitivity.
It is another object of the present invention to propose the preparation method of the electrochemical luminescence sensor of the detection dopamine.
Third object of the present invention is to propose the application of the electrochemical luminescence sensor.
Realize the technical solution of above-mentioned purpose of the present invention are as follows:
A kind of electrochemical luminescence sensor detecting dopamine, the electrochemical luminescence sensor includes electrode, the electricity
Pole includes substrate, composite layer and decorative layer, and the substrate is conductive material, and the composite layer is grown on the conductive substrates
It on surface, is polymerized by luminol and benzidine sulfate, the decorative layer is by modifying the tyrasamine oxidation in composite layer surface
Enzyme is constituted.
Due to the tyramine oxidase property of can choose catalysis oxidation dopamine, generated in-situ H2O2It can significant enhanced sensitivity Rumi
Tyramine oxidase is fixed on compound film electrode surface as dopamine by promise-benzidine sulfate electrochemical luminescence, the present invention
Recognition element, by the sensitivity knot of the selectivity of enzymic catalytic reaction and poly- (luminol-benzidine sulfate) electrochemical luminescence method
It is combined, constitutes the sensor of dopamine concentration measurement quick, sensitive, that selectivity is high.
The preparation method of electrochemical luminescence sensor of the present invention, comprising steps of
1) in phosphate buffered saline solution, luminol and benzidine sulfate solution are configured, using cyclic voltammetry, in conduction
Poly- (luminol-benzidine sulfate) composite layer is prepared in substrate,
2) above-mentioned electrode is taken out, it is dry, tyramine oxidase solution is configured with buffer solution, with cyclic voltammetry described
The fixed tyramine oxidase decorative layer in poly- (luminol-benzidine sulfate) the composite layer surface that step 1) obtains.
Preferably, in the step 1), the pH value of phosphate buffered saline solution is 5.5~8.5, in cyclic voltammetry preparation,
Using conductive substrates as working electrode, platinized platinum is to electrode, and saturated calomel electrode is reference electrode, and the conductive substrates are that FTO (mixes
The SnO of miscellaneous fluorine2Transparent conducting glass) or ITO (tin indium oxide) electro-conductive glass.
The pH value of phosphate buffered saline solution is smaller on the influence of poly- (luminol-benzidine sulfate) film properties, in institute of the present invention
Preparation can obtain poly- (luminol-benzidine sulfate) film in the range of the pH=5.5 of elaboration~8.5.
In the step 1), the molar concentration of luminol is 1~10mmol/L in buffer salt solution, and benzidine sulfate rubs
Your concentration is 1.5~15mmol/L.
It is highly preferred that the molar ratio of luminol and benzidine sulfate is 3:2~2:3.
Wherein, it in the step 1), when preparing poly- (luminol-benzidine sulfate) composite layer in conductive substrates, follows
The potential range of ring voltammetric scan is -0.2V~1.0V, and scanning circle number 30~100 encloses, 80~200mV/s of sweep speed.
Wherein, in the step 2), the buffer solution is the phosphate buffer solution that pH is 6.5~7.5, described slow
Rush the tyramine oxidase for being 0.05~2mg/mL containing concentration in solution.
Further, in the step 2), the potential range of cyclic voltammetry scan is -0.2V~0.6V, scanning circle number 10
~30 circles, 80~150mV/s of sweep speed.
The present invention also proposes the application of the electrochemical luminescence sensor.
Using the method for electrochemical luminescence sensor proposed by the present invention detection dopamine, the pH value of solution to be measured is adjusted
It is 6.8~7.2, under the conditions of being protected from light, the electrode of the electrochemical luminescence sensor is placed in solution to be measured, applies square wave electricity
Position, potential range are -0.5~-0.9V (vs.Ag/AgCl), the response signal of measuring electrode.
Preferably, square wave current potential can be selected with 0V, and the voltage of -0.5~-0.9V (vs.Ag/AgCl) two ladders is one
A circulation recycles 2~5 times.
Wherein, the pH value of solution to be measured is adjusted with phosphate-buffered salt or borax buffer solution.
Wherein, in solution to be measured the content of dopamine in 0.5nmol/L or more.
The beneficial effects of the present invention are:
Based on the method for electrochemical luminescence test dopamine concentration, made different from traditional using luminol or Polyluminol
For electrochemical luminescence sensor, luminol and benzidine sulfate are carried out electrochemical polymerization, utilize sulfuric acid biphenyl by present invention selection
The Photophysics of amine modulation enhancing luminol, and the identification substance for using tyramine oxidase as dopamine is modified in above-mentioned preparation
Poly- (luminol-benzidine sulfate) composite membrane on, the electrochemical luminescence sensor for detecting dopamine concentration is made.
Biologically active enzyme is used for electrochemiluminescence analysis by the present invention, can effectively solve electrochemical luminescence method
The problem of poor selectivity.The preparation of electrochemical sensor of the present invention and detection method are simple, high sensitivity, dopamine detection process
In do not influenced by uric acid and ascorbic acid etc., anti-interference is good.
Detailed description of the invention
Fig. 1 a is the AFM picture of poly- (luminol-benzidine sulfate) film prepared in embodiment of the present invention 1A)；Fig. 1 b is
Embodiment of the present invention 1B) in prepare tyramine oxidase-it is poly- (luminol-benzidine sulfate), i.e., the present invention in electrochemistry hair
The AFM picture of optical sensor；
Fig. 2 a is middle poly- (luminol-benzidine sulfate) film using cyclic voltammetry preparation of embodiment of the present invention 1A)
Reaction process curve；Fig. 2 b is embodiment of the present invention 1B) it is middle using cyclic voltammetry (luminol-poly- in inventive embodiments 1A)
Benzidine sulfate) the fixed tyramine oxidase of film surface reaction process curve；
Fig. 3 a detects electrochemical luminescence when different dopamine concentrations for electrochemical luminescence sensor in the embodiment of the present invention 2
Signal；Fig. 3 b is the electrochemical luminescence signals intensity of sensor in the embodiment of the present invention 2 with the variation of dopamine concentration and linear
Fit solution；
Fig. 4 is the anti-interference test result in the embodiment of the present invention 3, specially electrochemistry of the sensor in different solutions
Luminous signal intensity, wherein DA is dopamine, and AA is ascorbic acid, and UA is uric acid.
Fig. 5 is that the present invention proposes electrochemical sensor structural schematic diagram.In figure, 1 is conductive substrates, and 2 be composite layer, 3
For decorative layer.
Specific embodiment
The present invention is now illustrated with following most preferred embodiment, but is not intended to limit the scope of the invention.
Unless otherwise specified, means used in the examples are technological means commonly used in the art.
Embodiment 1:
In the present embodiment, the preparation method of electrochemical luminescence sensor includes the following steps
A the preparation of) poly- (luminol-benzidine sulfate) film
Phosphate buffered saline solution is configured, by changing 0.1mol/L NaH2PO4With 0.1mol/L Na2HPO4Aqueous solution
The pH value of proportion adjustment phosphate buffered saline solution is 5.7.Test result shows solution ph to poly- (luminol-sulfuric acid biphenyl
Amine) film properties influence it is smaller, poly- (luminol-benzidine sulfate) film can be obtained by preparing in the range of pH=5.5~8.5；It will
Luminol and benzidine sulfate, which are added in above-mentioned phosphate buffer solution, is configured to reaction solution, and specific concentration is the Shandong 8mmol/L
Minot and 12mmol/L benzidine sulfate select FTO electro-conductive glass to make substrate, using FTO as working electrode, platinized platinum be to electrode,
Saturated calomel electrode is reference electrode, is aggregated to above-mentioned luminol and benzidine sulfate using cyclic voltammetric electrochemical polymerization method
In FTO substrate, cyclic voltammetric parameter is set as scanning step 100mV/s, scanning range -0.2V~1.0V, and cycle-index 40 is gathered
The curve of conjunction process is as shown in Figure 2 a.It is dry by above-mentioned compound Membrane cleaning, poly- (luminol-benzidine sulfate) film is obtained,
Afm image is as shown in Figure 1a (coordinate unit of Fig. 1 is μm).
B) the fixation of tyramine oxidase
Phosphate buffered saline solution is configured, by changing 0.1mol/L NaH2PO4With 0.1mol/L Na2HPO4Aqueous solution
The pH value of proportion adjustment phosphate buffered saline solution is 7, with the phosphate buffered saline solution by tyramine oxidase with 0.1mg/mL's
Ratio is made into reaction solution.Using poly- (luminol-benzidine sulfate) film obtained in step A) for being grown in the surface FTO as substrate i.e.
Working electrode, platinized platinum are to electrode, and saturated calomel electrode is reference electrode, is consolidated above-mentioned tyramine oxidase using cyclic voltammetry
It is scheduled in substrate, specific cyclic voltammetric parameter is set as scanning step 100mV/s, scanning range -0.2V~0.6V, cycle-index
20, response curve is as shown in Figure 2 b.It is dry by sample clean, obtain complexes membrane, i.e. electrochemical luminescence sensor, AFM figure
As shown in Figure 1 b.
Electrode structure obtained such as Fig. 5, conductive substrates 1 are FTO electro-conductive glass, and composite layer 2 is by luminol and sulfuric acid
Benzidine is polymerized, and decorative layer 3 is made of the tyramine oxidase for modifying in composite layer surface.
Embodiment 2
The method that electrochemical luminescence sensor made from embodiment 1 is used to detect dopamine concentration includes the following steps:
Test carries out at room temperature, before test, phosphate buffered saline solution is configured, by changing 0.1mol/L
NaH2PO4With 0.1mol/L Na2HPO4The pH value of the proportion adjustment phosphate buffered saline solution of aqueous solution is 7, slow in the phosphoric acid
Rushing addition dopamine in salting liquid makes it distinguish concentration 0.5nmol/L, 1nmol/L, 10nmol/L and 20nmol/L, to implement
The electrochemical luminescence sensor prepared in example 1 is working electrode, and platinum filament is to electrode, and Ag/AgCl (saturation KCl) is reference electricity
Above-mentioned system is placed in the camera bellows of Weak-luminescence instrument by pole, applies following square wave current potential to the above system: (13s, 0V) and
(4s, -0.85V) (vs.Ag/AgCl), two square waves are a circulation, work four and recycle.It is right first in test process
The phosphate buffered saline solution of 0.1mol/L, pH=7 are tested, then that above-mentioned phosphate-buffered salt is molten to obtain blank signal
Liquid changes the dopamine solution of above-mentioned four kinds of concentration into, carries out the luminous test of optical electro-chemistry to the above system, obtains sample signal, most
The electrochemical luminescence intensity for obtaining sensor eventually is that sample signal subtracts blank signal.Fig. 3 a show the electrochemical luminescence and passes
Sensor is to the dopamine response signal of various concentration, and with the increase of dopamine concentration, luminous signal also enhances therewith, the sensing
Device has reached 0.5nmol/L to the detection limit of dopamine.Dopamine concentration and electrochemical luminescence signals are mapped, line of going forward side by side
It is fitted (Fig. 3 b, coefficient R=0.9991), the ordinate of Fig. 3 is electrochemical luminescence intensity in test process, in 1nmol/L
Good linear relationship is presented in the luminous intensity and dopamine concentration of~20nmol/L range inner sensor.
Embodiment 3
Electrochemical luminescence sensor proposed by the present invention exists for detecting dopamine in high concentration uric acid and ascorbic acid
Under the conditions of selectivity test it is as follows:
Test carries out at room temperature, before test, phosphate buffered saline solution is configured, by changing 0.1mol/L
NaH2PO4With 0.1mol/L Na2HPO4The pH value of the proportion adjustment phosphate buffered saline solution of aqueous solution is 7, is added in the solution
1nmol/L dopamine, 100nmol/L uric acid and 100nmol/L ascorbic acid are made into interference test solution, in embodiment 1
The electrochemical luminescence sensor of preparation is working electrode, and platinum filament is to electrode, and Ag/AgCl (saturation KCl) is reference electrode, will be upper
The system of stating is placed in the camera bellows of Weak-luminescence instrument, applies following square wave current potential: (13s, 0V) and (4s, -0.85V) to it
(vs.Ag/AgCl), four circulations are tested.We test 0.1mol/L, the phosphate buffered saline solution of pH=7 first, with
Blank signal is obtained, the interference configured before then changing above-mentioned phosphate buffered saline solution into tests solution, to above-mentioned system
It is tested, obtains sample signal, the electrochemical luminescence intensity of finally obtained sensor is that sample signal subtracts blank signal.
Fig. 4 reflects interference test result, it is found that under conditions of 100 times of dopamine concentrations, uric acid and Vitamin C
The electrochemical luminescence signals of acid are well below dopamine, and two kinds of interference substances can be ignored, it follows that prepared electrification
Learning luminescence sensor has selectivity well to dopamine detection.
Above embodiment be only preferred embodiments of the present invention will be described, not to the scope of the present invention into
Row limits, and without departing from the spirit of the design of the present invention, this field ordinary engineering and technical personnel is to technical side of the invention
The all variations and modifications that case is made, should fall within the scope of protection determined by the claims of the present invention.
Claims (6)
1. a kind of electrochemical luminescence sensor for detecting dopamine, the electrochemical luminescence sensor includes electrode, the electrode
Including substrate, composite layer and decorative layer, which is characterized in that the substrate is conductive material, and the composite layer is grown on institute
It states on the surface of substrate, is polymerized by luminol and benzidine sulfate, the decorative layer is by modifying in composite layer surface
Tyramine oxidase is constituted；
The electrochemical luminescence sensor is prepared by following steps:
1) in phosphate buffered saline solution, luminol and benzidine sulfate solution are configured, using cyclic voltammetry, in conductive substrates
It is upper to prepare poly- (luminol-benzidine sulfate) composite layer, wherein the pH value of phosphate buffered saline solution is 5.5~8.5, circulation
In voltammetry preparation, using conductive substrates as working electrode, platinized platinum is to electrode, and saturated calomel electrode is reference electrode, described to lead
Electric substrate is FTO or ITO electro-conductive glass；The molar concentration of luminol is 1~10mmol/L, benzidine sulfate in buffer salt solution
Molar concentration be 1.5~15mmol/L；
2) above-mentioned electrode is taken out, it is dry, tyramine oxidase solution is configured with buffer solution, with cyclic voltammetry in the step
1) the fixed tyramine oxidase decorative layer in poly- (luminol-benzidine sulfate) the composite layer surface obtained；The buffer solution is
The phosphate buffer solution that pH is 6.5~7.5 aoxidizes in the buffer solution containing the tyrasamine that concentration is 0.05~2mg/mL
Enzyme.
2. electrochemical luminescence sensor according to claim 1, which is characterized in that in the step 1), in conductive substrates
Upper the potential range of cyclic voltammetry scan is -0.2V~1.0V when preparing poly- (luminol-benzidine sulfate) composite layer, is swept
It retouches circle number 30~100 to enclose, 80~200mV/s of sweep speed.
3. electrochemical luminescence sensor according to claim 1 or 2, which is characterized in that in the step 2), cyclic voltammetric
The potential range of scanning is -0.2V~0.6V, and scanning circle number 10~30 encloses, 80~150mV/s of sweep speed.
4. the method for any one of the application claims 1 to 3 electrochemical luminescence sensor detection dopamine, which is characterized in that
The pH value of solution to be measured is adjusted to 6.8~7.2, under the conditions of being protected from light, the electrode of the electrochemical luminescence sensor is placed in
In solution to be measured, apply square wave current potential, potential range is -0.5~-0.9Vvs.Ag/AgCl, the response signal of measuring electrode.
5. the method for electrochemical luminescence sensor detection dopamine according to claim 4, which is characterized in that use phosphoric acid buffer
Salt or borax buffer solution adjust the pH value of solution to be measured.
6. the method for electrochemical luminescence sensor detection dopamine according to claim 4 or 5, which is characterized in that be measured molten
The content of dopamine is in 0.5nmol/L or more in liquid.
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| CN103592292A (en) * | 2013-10-28 | 2014-02-19 | 江苏大学 | Method for rapidly sensitively determining dopamine |
| CN105115961A (en) * | 2015-08-07 | 2015-12-02 | 上海师范大学 | Method for preparing electrochemical luminescence sensor made of nano-composites |
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| CN101493453A (en) * | 2008-12-30 | 2009-07-29 | 上海纳米技术及应用国家工程研究中心有限公司 | Thin layer detecting pool for detecting dopamine and method for making same |
| CN102706864B (en) * | 2012-05-25 | 2015-04-22 | 苏州大学 | Luminol-aniline copolymerization modified electrode and preparation method thereof |
| CN103149267B (en) * | 2013-02-06 | 2015-02-18 | 河南省科学院高新技术研究中心 | Electrochemical biosensor for detecting dopamine and its preparation method |
| CN103675076A (en) * | 2013-12-13 | 2014-03-26 | 济南大学 | Preparation method and application of electrochemical aptamer sensor for detecting dopamine (DA) |
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| CN105115961A (en) * | 2015-08-07 | 2015-12-02 | 上海师范大学 | Method for preparing electrochemical luminescence sensor made of nano-composites |
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| Title |
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| An electrochemiluminescent biosensor for dopamine detection using a poly(luminol–benzidine sulfate) electrode modified by tyramine oxidase;Yanjie Wang等;《New J. Chem.》;20170104;第41卷(第4期);第1591-1597页 |
| Electrochemiluminescence Characterization of Poly(luminol-benzidine) Composite Films and Their Analytical Application;Guixin Li等;《Electroanalysis》;20090325;第21卷(第7期);第845-852页 |
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