CN106248748A - A kind of acetylcholinesterasebiosensor biosensor and application thereof - Google Patents
A kind of acetylcholinesterasebiosensor biosensor and application thereof Download PDFInfo
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- NUJOXMJBOLGQSY-UHFFFAOYSA-N manganese dioxide Inorganic materials O=[Mn]=O NUJOXMJBOLGQSY-UHFFFAOYSA-N 0.000 claims abstract description 48
- 239000000575 pesticide Substances 0.000 claims abstract description 36
- 238000001514 detection method Methods 0.000 claims abstract description 26
- 239000000463 material Substances 0.000 claims abstract description 24
- 102000012440 Acetylcholinesterase Human genes 0.000 claims abstract description 21
- 108010022752 Acetylcholinesterase Proteins 0.000 claims abstract description 21
- 229940022698 acetylcholinesterase Drugs 0.000 claims abstract description 21
- DUEPRVBVGDRKAG-UHFFFAOYSA-N carbofuran Chemical compound CNC(=O)OC1=CC=CC2=C1OC(C)(C)C2 DUEPRVBVGDRKAG-UHFFFAOYSA-N 0.000 claims abstract description 17
- RLBIQVVOMOPOHC-UHFFFAOYSA-N parathion-methyl Chemical group COP(=S)(OC)OC1=CC=C([N+]([O-])=O)C=C1 RLBIQVVOMOPOHC-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229910021397 glassy carbon Inorganic materials 0.000 claims abstract description 15
- 239000002060 nanoflake Substances 0.000 claims abstract description 15
- 229920001661 Chitosan Polymers 0.000 claims abstract description 12
- 239000002114 nanocomposite Substances 0.000 claims abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 12
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims abstract description 6
- 239000012286 potassium permanganate Substances 0.000 claims abstract description 6
- 229910002621 H2PtCl6 Inorganic materials 0.000 claims abstract description 5
- 229910002666 PdCl2 Inorganic materials 0.000 claims abstract description 5
- 229910000033 sodium borohydride Inorganic materials 0.000 claims abstract description 5
- 239000012279 sodium borohydride Substances 0.000 claims abstract description 5
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims abstract description 5
- 239000001509 sodium citrate Substances 0.000 claims abstract description 5
- 229920002565 Polyethylene Glycol 400 Polymers 0.000 claims abstract description 4
- PIBWKRNGBLPSSY-UHFFFAOYSA-L palladium(II) chloride Chemical compound Cl[Pd]Cl PIBWKRNGBLPSSY-UHFFFAOYSA-L 0.000 claims abstract description 4
- JLFNLZLINWHATN-UHFFFAOYSA-N pentaethylene glycol Chemical compound OCCOCCOCCOCCOCCO JLFNLZLINWHATN-UHFFFAOYSA-N 0.000 claims abstract description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Substances [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 48
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 238000002484 cyclic voltammetry Methods 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 9
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 8
- 239000012498 ultrapure water Substances 0.000 claims description 8
- -1 mercaptoacetyl choline Chemical compound 0.000 claims description 7
- 229910052799 carbon Inorganic materials 0.000 claims description 5
- 239000002131 composite material Substances 0.000 claims description 5
- 238000001548 drop coating Methods 0.000 claims description 5
- 230000003466 anti-cipated effect Effects 0.000 claims description 4
- 229960001231 choline Drugs 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 238000010992 reflux Methods 0.000 claims description 4
- 230000001629 suppression Effects 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 3
- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical class Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 229910052697 platinum Inorganic materials 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 2
- 238000005498 polishing Methods 0.000 claims description 2
- 238000004506 ultrasonic cleaning Methods 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims 1
- 230000006641 stabilisation Effects 0.000 claims 1
- 238000011105 stabilization Methods 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 10
- 102000004190 Enzymes Human genes 0.000 abstract description 7
- 108090000790 Enzymes Proteins 0.000 abstract description 7
- 229940088598 enzyme Drugs 0.000 abstract description 7
- 239000000447 pesticide residue Substances 0.000 abstract description 7
- 238000005516 engineering process Methods 0.000 abstract description 4
- 238000000835 electrochemical detection Methods 0.000 abstract description 3
- 230000000977 initiatory effect Effects 0.000 abstract description 3
- 238000011896 sensitive detection Methods 0.000 abstract description 3
- 238000010521 absorption reaction Methods 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 abstract description 2
- 230000005518 electrochemistry Effects 0.000 abstract description 2
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 6
- 230000003197 catalytic effect Effects 0.000 description 6
- 229960004373 acetylcholine Drugs 0.000 description 4
- GFFIJCYHQYHUHB-UHFFFAOYSA-N 2-acetylsulfanylethyl(trimethyl)azanium Chemical compound CC(=O)SCC[N+](C)(C)C GFFIJCYHQYHUHB-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 229910000990 Ni alloy Inorganic materials 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- PCLURTMBFDTLSK-UHFFFAOYSA-N nickel platinum Chemical compound [Ni].[Pt] PCLURTMBFDTLSK-UHFFFAOYSA-N 0.000 description 3
- 229910052763 palladium Inorganic materials 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000000956 alloy Substances 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
- 238000009833 condensation Methods 0.000 description 2
- 230000005494 condensation Effects 0.000 description 2
- 229960000935 dehydrated alcohol Drugs 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000005611 electricity Effects 0.000 description 2
- 229960004756 ethanol Drugs 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 229910045601 alloy Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- CVXBEEMKQHEXEN-UHFFFAOYSA-N carbaryl Chemical compound C1=CC=C2C(OC(=O)NC)=CC=CC2=C1 CVXBEEMKQHEXEN-UHFFFAOYSA-N 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000003859 hyphenated technique Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 231100000567 intoxicating Toxicity 0.000 description 1
- 230000002673 intoxicating effect Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002398 materia medica Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- JRTYPQGPARWINR-UHFFFAOYSA-N palladium platinum Chemical compound [Pd].[Pt] JRTYPQGPARWINR-UHFFFAOYSA-N 0.000 description 1
- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 1
- 239000010970 precious metal Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of acetylcholinesterasebiosensor biosensor and application thereof, belong to technical field of electrochemical detection.This biosensor is obtained by the following steps: first, with KMnO4、CTAB、PdCl2、PEG400、H2PtCl6, sodium citrate and sodium borohydride be initiation material, use water bath Method to prepare MnO2Nanoflakes nano composite material and Pd Pt@MnO2Nanoflakes nano composite material；Secondly, use physical absorption that acetylcholinesterase (AChE) is fixed on Pd Pt@MnO with the fixing acetylcholinesterase of chitosan (CS)2The glassy carbon electrode surface modified is built into electrochemica biological sensor.The present invention senses combination technology by using electrochemistry with enzyme, it is achieved that to parathion-methyl and the highly sensitive detection of Furadan pesticide, and required sample is few, and the detection time is short, highly sensitive, low cost, it is adaptable to the analysis of pesticide residues and detection.
Description
Technical field
The present invention relates to a kind of acetylcholinesterasebiosensor biosensor and application thereof, be specifically related to load based on platinum-nickel alloys
The electrochemica biological sensor of manganese dioxide nano-composite material and application thereof, belong to technical field of electrochemical detection.
Background technology
Pesticide is as the special means of production a kind of in agricultural production process, and it can be with eliminating pest, increase yield, guarantee
Harvest, moreover it is possible to prevent and control human infectious disease.But the food-safety problem that pesticide uses initiation in a large number also becomes increasingly conspicuous.Pesticide
Entering human body by contaminated agricultural product, people will produce intoxicating phenomenon, and some harmful elements may cause human body cell canceration,
Even also affect offspring healthy.Therefore, in order to improve and ensure food quality and life security, the Gao Ling to pesticide residues
Quick detection became in recent years focus of attention both at home and abroad.At present, Detecting Pesticide gas chromatogram, high performance liquid chromatography, color
The technology such as spectrum-mass spectrometric hyphenated technique, capillary electrophoresis, surface enhanced raman spectroscopy method, immunoassay, biosensor are answered
Use in the real work of Detecting Pesticide.But due to gas chromatogram, high performance liquid chromatography and chromatograph-mass spectrometer coupling technology,
Expensive equipment and be difficult to realize miniaturization, the pretreatment process of sample is relatively complicated；Capillary electrophoresis then capillary diameter
Little, light is short out, poor reproducibility；Surface enhanced raman spectroscopy method repeatability and poor stability.Therefore simplicity, quickly, accurately is built
With efficient pesticide residue detection method, the safe and sanitary caused and environmental pollution are effectively solved to be exceeded standard by pesticide residues
Become current focus.
Electrochemica biological sensor occupies an important position in current sensor, its have highly sensitive, instrument from
Dynamicization degree is high, sample consumes less, response time is short, easy and simple to handle, with low cost, is converted to directly with its detection signal while of this
See the concentration value of readability, it is simple to layman uses, and has the most been widely used in biomedicine, environmental science, materia medica
And the field such as Food Science.Enzyme method also result in the extensive concern of people in the detection of pesticide residues, mainly due to
Possess simple to operate, selectivity good, highly sensitive, response time fast and the above advantage such as the miniaturization of detecting instrument.
Palladium load nano material of manganese dioxide is the most delivered by substantial amounts of and refer to a lot of and chemical phase widely
The field closed, palladium belongs to precious metal element, the electro catalytic activity having had and electric conductivity, and in addition also to have palladium preferable for platinum
Electro catalytic activity and dispersibility, therefore make alloy by palladium platinum and load on manganese dioxide simultaneously, this alloy material tool of preparation
There is higher electro catalytic activity, and acetylcholinesterase can be fixed and then for detection by quantitative with this kind of material of first passage
The pesticide such as parathion-methyl and Furadan, reach to required by Pesticides Testing highly sensitive, selectivity good, the detection time is short
Demand.
Summary of the invention
Electro catalytic activity is high, highly sensitive, selectivity is good, the detection time is short and has to it is an object of the invention to provide one
Help the fixing electrochemical biosensor based on platinum-nickel alloys load manganese dioxide nano-composite material of acetylcholinesterase
Device and application thereof.
For achieving the above object, the present invention uses following technical proposals: multiple based on platinum-nickel alloys load manganese dioxide nano
The preparation of the acetylcholinesteraseelectrochemistry electrochemistry biosensor of condensation material, it comprises the following steps: first, with KMnO4、CTAB、
PEG400 and sodium citrate, sodium borohydride, PdCl2And H2PtCl6For initiation material, water bath Method is used to prepare Pd-Pt@MnO2
Nano composite material.Use drop-coating by the Pd-Pt@MnO of preparation2Modify glassy carbon electrode surface；Secondly, with chitosan (CS)
Fixing acetylcholinesterase (AChE), uses physical absorption that acetylcholinesterase (AChE) is fixed on Pd-Pt@MnO2Modify glass
Carbon electrodes, based on Pd-Pt@MnO2Good chemical property, is built into electrochemica biological sensor and is applied to methyl
Quick, the Sensitive Detection of the pesticide such as parathion and Furadan, its building process and detection mechanism:
Acetylcholine ester substrate for enzymatic activity acetylthiocholine hydrolyzes, and generates product thiocholine and acetic acid.Thiocholine
Under specific potential the most oxidized, produce electric current.Under conditions of concentration of substrate is certain, current signal size and biosensor
Catalytic response relevant.Under having pesticide existence condition, pesticide and hydroxy combining in the activity of acetylcholinesterase, generate sulfur
Phosphorylated acetylcholinesterase for acetylcholine catalytically inactive:
Acetylcholinesterase is catalyzed activity to substrate to be suppressed by pesticide, the thiocholine lowering of concentration of generation, electricity
Stream reduces. and along with the increase of pesticide concentration, the suppression ratio of enzyme electrode is increased by it, according to following formula calculating percent inhibition [15]:
Suppression ratio=[(I0 I1)/I0] × 100% (1)
(1), in formula, I0 refers to the stable state electricity that certain density acetylthiocholine is produced by acetylcholine ester enzyme sensor
Stream, refers to the steady-state current that same concentration acetylthiocholine is produced after pesticide suppresses by acetylcholine ester enzyme sensor, logical
Cross the current signal i.e. oxidation peak size compared before and after enzyme level, it is achieved to parathion-methyl, the agriculture such as sevin and Furadan
The detection by quantitative of medicine.
The acetylcholinesterasebiosensor biosensor of the present invention obtains via following steps:
(1)Pd-Pt@MnO2The preparation of-nanoflakes nano composite material
①MnO2The preparation of-nanoflakes material
911mg cetyl trimethylammonium bromide is dissolved in the ultra-pure water of 25ml, is heated to reflux to temperature being maintained at 140 DEG C
Time, it is subsequently added into KMnO4Its quality is that 79.02mg is dissolved in the ultra-pure water of 25mL, backflow when temperature constant is maintained at 140 DEG C
8 to 10 minutes, color from purple became dark-brown, was cooled to room temperature, washes three times with ethanol, is settled to 10mL with water, i.e. obtains
MnO2-nanoflakes nano composite material；
②Pd-Pt@MnO2The preparation of-nanoflakes composite
Take the MnO that 1. 5mL prepares2Nanoflakes material, adds 0.5mL 10mM PdCl2, 0.5mL10mM
H2PtCl6, 0.1mL PEG400 and 1.0mL 10mM sodium citrate stirring and evenly mixing, it is eventually adding the reaction of 3mL15mM sodium borohydride
4h, washes three times afterwards with water and is centrifuged, be settled to 5mL, i.e. obtain Pd-Pt@MnO2Composite；
(2) glassy carbon electrode surface pretreatment
By the glass-carbon electrode Glassy carbon electrode Al of a diameter of 3mm2O3Stick with paste polishing and become minute surface, depend on
Secondary ethanol and redistilled water ultrasonic cleaning, be positioned over natural drying in air, then in the PBS solution of pH 7.4 in+
Scan 300s under 1.75V constant potential, then at scan round between+0.3V～+1.25V and+0.3V～-1.3V 20 times, treat electric current
After Wen Ding, with secondary water rinse electrode and in atmosphere natural drying standby；
(3) structure at electrochemical biosensor interface
Take the Pd-Pt@MnO that 6uL obtains2Hanging drop is applied to the glassy carbon electrode surface anticipated, and puts at room temperature
It is dried, i.e. obtains Pd-Pt@MnO2Modified glassy carbon electrode, is designated as Pd-Pt@MnO2/GCE；Finally treat that Pd-Pt@MnO2 modifies glass carbon
Followed by electrode surface drop coating 4.5 μ L acetylcholinesterase (AChE) and chitosan (CS) mixture after electrode is dry, wherein: second
Acetylcholinesterase is 3 μ L and chitosan is 1.5 μ L, is dried overnight under 4 DEG C of environment, i.e. obtains AChE-CS/Pd-Pt@MnO2/
GCE；
(4) set up parathion-methyl and the standard curve of Furadan Pesticides Testing
AChE-CS/Pd-Pt@MnO2/ GCE is as working electrode, saturated calomel electrode (saturated calomel
Electrode) as reference electrode, platinum electrode (platinum wire electrode) conduct, will during mensuration pesticide to electrode
Working electrode is placed in the middle suppression 4min cyclic voltammetry scan containing variable concentrations parathion-methyl and variable concentrations Furadan
20 times, after background current is stable, incubation 10min, record at 25 DEG C in mercaptoacetyl choline (ATCl) solution of 2mmol/L
Cyclic voltammogram produced by each of which, current value, with the change that the variable concentrations of pesticide is rule, draws standard curve also
Determine optimum linear scope and the detection limit of pesticide；The range of linearity of parathion-methyl Pesticides Testing is (10-13) mol/L～
(10-8) mol/L, its detection is limited to (0.35 × 10-13)mol/L；The range of linearity of Furadan Pesticides Testing is (10-12)mol/L
～(10-8) mol/L, its detection is limited to (0.3 × 10-12)mol/L。
The acetylcholinesterasebiosensor biosensor of the present invention is to the application in parathion-methyl and Furadan Pesticides Testing.
The beneficial effects of the present invention is:
1, the method utilizing water-bath prepares Pd-Pt@MnO2Nano composite material, its electro catalytic activity with excellence and life
The thing compatibility；
2, constructed electrochemica biological sensor, be successfully realized parathion-methyl and Furadan pesticide is highly sensitive
Detection, wherein the range of linearity of parathion-methyl Pesticides Testing is (10-13) mol/L～(10-8) mol/L, its detection is limited to
(0.35×10-13)mol/L；The range of linearity of Furadan Pesticides Testing is (10-12) mol/L～(10-8) mol/L, its detection limit
For (0.3 × 10-12)mol/L；
3, the present invention is based on the pesticide inhibitory action to enzyme modified electrode, it is achieved that the mesh of Electrochemical Detection pesticide residues
, needed for the method, sample is few, the detection time is short and sensitivity is higher, it is adaptable to the analysis of pesticide residues and detection.
Accompanying drawing explanation
Fig. 1 is the cyclic voltammogram of several different materials electrode, all occurs in that an irreversible oxidation peak in figure:
a.AChE-CS/GCE b.AChE-CS/MnO2-nanoflakes/GCE c.AChE-CS/Pd-Pt@MnO2/GCE。
The line of Fig. 2 black is electrochemica biological sensor electrode Pd-Pt@MnO2/ AChE-CS/GCE is applied to detection difference
The DPV figure of concentration parathion-methyl.
The line of Fig. 3 black is electrochemica biological sensor electrode Pd-Pt@MnO2/ AChE-CS/GCE is applied to detection difference
The i-t curve of concentration Furadan pesticide.
The line of Fig. 4 black is electrochemica biological sensor electrode Pd-Pt@MnO2/ AChE-CS/GCE is applied to detection difference
The standard curve of concentration parathion-methyl.
The line of Fig. 5 black is electrochemica biological sensor Pd-Pt@MnO2/ AChE-CS/GCE is applied to detect variable concentrations
The standard curve of Furadan pesticide.
Detailed description of the invention
In embodiment acetylcholinesterasebiosensor biosensor prepare via step with described in Summary.
Embodiment one:
First, the Pd-Pt@MnO of good dispersion is prepared2Nano composite material: 911mg cetyl trimethylammonium bromide is molten
In the ultra-pure water of 25mL, when being heated to reflux being maintained at 140 DEG C, it is subsequently added into KMnO4Its quality is that 79.02mg is dissolved in 25mL's
Ultra-pure water, backflow 8 to 10 minutes when temperature constant is maintained at 140 DEG C, color from purple becomes dark-brown, cools down room temperature, uses
Dehydrated alcohol is washed three times, is settled to 10mL (being equivalent to 6mg/mL) with water, i.e. obtains MnO2-nanoflakes material.Take 5mL system
The MnO obtained2-nanoflakes material, adds 0.5mL 10mM PdCl2, 0.5mL 10mM H2PtCl6, 0.1mLPEG400 and
1.0mL10mM sodium citrate stirring and evenly mixing, is eventually adding 3mL 15mM sodium borohydride reaction 4h, washes with water afterwards three times, centrifugal,
It is settled to 5mL, i.e. obtains Pd-Pt@MnO2Composite.
Take 6uL Pd-Pt obtained above@MnO2Hanging drop is applied to the glass-carbon electrode GCE surface anticipated, and is placed on
It is dried under room temperature, i.e. obtains Pd-Pt@MnO2Modified glassy carbon electrode, is designated as Pd-Pt@MnO2/GCE；Last at the most dry Pd-Pt@
MnO2The glassy carbon electrode surface drop coating 4.5 μ L acetylcholinesterase (AChE) modified and chitosan (CS) mixture, at 4 DEG C of environment
In be dried overnight, i.e. obtain AChE-CS/Pd-Pt@MnO2/GCE；
Select 10-13Mol/L～10-8The parathion-methyl pesticide of mol/L concentration range is at PBS (0.1mol/L pH 7.4)
Middle cyclic voltammetry scan 20 times, after background current is stable, in the mercaptoacetyl choline ATCl solution of 2mmol/L at 25 DEG C
Educate temperature 10min, cyclic voltammogram (as shown in Figure 2) produced by record each of which.Carry out quantitative determining (such as Fig. 2 institute with DPV
Show), Criterion curve (as shown in Figure 4), determine that the detection of this electrochemica biological sensor is limited to 0.35 × 10-13mol/L。
Embodiment two:
First, the Pd-Pt@MnO of good dispersion is prepared2Nano composite material: 911mg cetyl trimethylammonium bromide is molten
In the ultra-pure water of 25mL, be heated to reflux to temperature be maintained at 140 DEG C time, be subsequently added into KMnO4Its quality is that 79.02mg is dissolved in
In the ultra-pure water of 25mL 140 DEG C, backflow 8 to 10 minutes when temperature constant is maintained at 140 DEG C, color from purple becomes dark brown
Color, cools down room temperature, washes three times with dehydrated alcohol, be settled to 10mL (being equivalent to 7mg/mL) with water, i.e. obtain Pd-Pt MnO2Multiple
Condensation material.
Take the Pd-Pt@MnO that 6uL obtains2Hanging drop is applied to the glass-carbon electrode GCE surface anticipated, and is placed on room temperature
Lower dry, i.e. obtain Pd-Pt@MnO2Modified glassy carbon electrode, is designated as Pd-Pt@MnO2/GCE；Last at the most dry Pd-Pt@MnO2
The glassy carbon electrode surface drop coating 4.5 μ L acetylcholinesterase (AChE) modified and chitosan (CS) mixture, dry in 4 DEG C of environment
Dry, overnight, i.e. obtain AChE-CS/Pd-Pt@MnO2/GCE；
Select 10-12Mol/L～10-8The Furadan pesticide of mol/L concentration range is used in PBS (0.1mol/L pH7.4)
Cyclic voltammetry scan 20 times, after background current is stable, educates temperature at 25 DEG C in the mercaptoacetyl choline ATCl solution of 2mmol/L
10min, records cyclic voltammogram (as shown in Figure 1) produced by each of which.Carry out quantitative determining (such as Fig. 3 institute with i-t curve
Show), Criterion curve (as shown in Figure 5), determine that the detection of this electrochemica biological sensor is limited to 0.3 × 10-12mol/L。
Claims (2)
1. an acetylcholinesterasebiosensor biosensor, it is characterised in that this acetylcholinesterasebiosensor biosensor is via following step
Suddenly obtain:
(1)Pd-Pt@MnO2The preparation of-nanoflakes nano composite material
①MnO2The preparation of-nanoflakes material
911mg cetyl trimethylammonium bromide is dissolved in the ultra-pure water of 25ml, be heated to reflux to temperature be maintained at 140 DEG C time,
It is subsequently added into KMnO4Its quality is that 79.02mg is dissolved in the ultra-pure water of 25mL, when temperature constant is maintained at 140 DEG C backflow 8 to
10 minutes, color from purple became dark-brown, was cooled to room temperature, washes three times with ethanol, is settled to 10mL with water, i.e. obtains
MnO2-nanoflakes nano composite material；
②Pd-Pt@MnO2The preparation of-nanoflakes composite
Take the MnO that 1. 5mL prepares2Nanoflakes material, adds 0.5mL 10mM PdCl2, 0.5mL10mM H2PtCl6,
0.1mL PEG400 and 1.0mL 10mM sodium citrate stirring and evenly mixing, be eventually adding 3mL15mM sodium borohydride reaction 4h, use afterwards
Wash three times to be centrifuged, be settled to 5mL, i.e. obtain Pd-Pt@MnO2Composite；
(2) glassy carbon electrode surface pretreatment
By the glass-carbon electrode Glassy carbon electrode Al of a diameter of 3mm2O3Stick with paste polishing and become minute surface, use successively
Ethanol and redistilled water ultrasonic cleaning, be positioned over natural drying in air, then in+1.75V in the PBS solution of pH 7.4
Scan 300s under constant potential, then at scan round between+0.3V～+1.25V and+0.3V～-1.3V repeatedly, treat current stabilization
After, with secondary water rinse electrode and in atmosphere natural drying standby；
(3) structure at electrochemical biosensor interface
Take the Pd-Pt@MnO that 6uL obtains2Hanging drop is applied to the glassy carbon electrode surface anticipated, and puts and is dried at room temperature for,
I.e. obtain Pd-Pt@MnO2Modified glassy carbon electrode, is designated as Pd-Pt@MnO2/GCE；Finally treat that Pd-Pt@MnO2 modified glassy carbon electrode is done
After followed by electrode surface drop coating 4.5 μ L acetylcholinesterase and chitosan compound, wherein: acetylcholinesterase is 3 μ L
It is 1.5 μ L with chitosan, is dried overnight under 4 DEG C of environment, i.e. obtain AChE-CS/Pd-Pt@MnO2/GCE；
(4) set up parathion-methyl and the standard curve of Furadan Pesticides Testing
AChE-CS/Pd-Pt@MnO2/ GCE is as working electrode, and saturated calomel electrode is as reference electrode, and platinum electrode is as right
Electrode, is placed in the middle suppression containing variable concentrations parathion-methyl and variable concentrations Furadan by working electrode when measuring pesticide
4min is with cyclic voltammetry scan repeatedly, after background current is stable, warm at 25 DEG C in the mercaptoacetyl choline solution of 2mmol/L
Educating 10min, cyclic voltammogram produced by record each of which, current value, with the change that the variable concentrations of pesticide is rule, is painted
Standard curve processed also determines that the optimum linear scope of pesticide and detection limit；The range of linearity of parathion-methyl Pesticides Testing is (10-13) mol/L～(10-8) mol/L, its detection is limited to (0.35 × 10-13)mol/L；The range of linearity of Furadan Pesticides Testing is
(10-12) mol/L～(10-8) mol/L, its detection is limited to (0.3 × 10-12)mol/L。
2. the acetylcholinesterasebiosensor biosensor described in claim 1 is in parathion-methyl and Furadan Pesticides Testing
Application.
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