CN106248748B - A kind of acetylcholinesterasebiosensor biosensor and its application - Google Patents
A kind of acetylcholinesterasebiosensor biosensor and its application Download PDFInfo
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- CN106248748B CN106248748B CN201610584066.9A CN201610584066A CN106248748B CN 106248748 B CN106248748 B CN 106248748B CN 201610584066 A CN201610584066 A CN 201610584066A CN 106248748 B CN106248748 B CN 106248748B
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- 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
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- 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
Abstract
The present invention relates to a kind of acetylcholinesterasebiosensor biosensor and its applications, belong to technical field of electrochemical detection.The biosensor is obtained by the following steps：First, with KMnO4、CTAB、PdCl2、PEG400、H2PtCl6, sodium citrate and sodium borohydride be starting material, MnO is prepared using water bath Method2- nanoflakes nanocomposites and Pd-Pt@MnO2- nanoflakes nanocomposites；Secondly, acetylcholinesterase (AChE) is fixed on by Pd-Pt@MnO using physical absorption with chitosan (CS) fixed acetylcholinesterase2The glassy carbon electrode surface of modification is built into electrochemica biological sensor.The present invention senses combination technology by using electrochemistry and enzyme, realizes the highly sensitive detection to parathion-methyl and Furadan pesticide, and required sample is few, and detection time is short, high sensitivity, and low cost is suitable for the analysis and detection of pesticide residue.
Description
Technical field
The present invention relates to a kind of acetylcholinesterasebiosensor biosensor and its applications, and in particular to is loaded based on platinum-nickel alloys
The electrochemica biological sensor of manganese dioxide nano-composite material and its application, belong to technical field of electrochemical detection.
Background technology
For pesticide as a kind of special means of production in agricultural production process, it can be with eliminating pest, increase yield, guarantee
Harvest, moreover it is possible to prevent and control human infectious disease.But pesticide is largely also become increasingly conspicuous using the food-safety problem of initiation.Pesticide
Entering human body by contaminated agricultural product, people will generate intoxicating phenomenon, some harmful elements may cause human body cell canceration,
Even also affect offspring's health.Therefore, in order to improve and ensure food quality and life security, to the Gao Ling of pesticide residue
Quick detection becomes domestic and international focus of attention in recent years.Currently, Detecting Pesticide gas-chromatography, high performance liquid chromatography, color
The technologies such as spectrum-mass spectrometric hyphenated technique, Capillary Electrophoresis, Surface enhanced Raman spectroscopy method, immunoassay, biosensor are answered
In the real work for using Detecting Pesticide.But due to gas-chromatography, high performance liquid chromatography and chromatograph-mass spectrometer coupling technology,
It expensive equipment and is not easy to realize micromation, the pretreatment process of sample is relatively complicated；Capillary Electrophoresis then capillary diameter
Small, light path is short, poor reproducibility；Surface enhanced Raman spectroscopy method reproducibility and stability are poor.Therefore it is easy, quick, accurate to build
With efficient pesticide residue detection method, effective solution safe and healthy and environmental pollution caused by excessive pesticide residues is
As current focus.
Electrochemica biological sensor occupies an important position in current sensor, certainly with high sensitivity, instrument
Dynamicization degree is high, sample consumption is few, the response time is short, easy to operate, of low cost, and with this, its detection signal is converted to directly simultaneously
The concentration value for seeing readability uses convenient for layman, has been widely used in biomedicine, environmental science, materia medica at present
And the fields such as Food Science.Enzyme method also results in the extensive concern of people in the detection of pesticide residue, mainly due to
Have that easy to operate, the good, high sensitivity of selectivity, response time be fast and the above advantage such as the micromation of detecting instrument.
Palladium load nano material of manganese dioxide has been delivered and has widely been referred to many with chemical phase by a large amount of at present
The field of pass, palladium belong to precious metal element, the electro catalytic activity and electric conductivity having had, and in addition to this platinum also has palladium preferable
Electro catalytic activity and dispersibility, therefore palladium platinum is made into alloy while being loaded on manganese dioxide, this alloy material tool of preparation
There is stronger electro catalytic activity, and can be with such material of first passage to fix acetylcholinesterase and then for quantitatively detecting
The pesticides such as parathion-methyl and Furadan, reach to required by Pesticides Testing high sensitivity, selectivity is good, detection time is short etc.
Demand.
Invention content
The purpose of the present invention is to provide a kind of electro catalytic activity height, high sensitivity, selectivity is good, detection time is short and has
Help the fixed electrochemical biosensor that manganese dioxide nano-composite material is loaded based on platinum-nickel alloys of acetylcholinesterase
Device and its application.
To achieve the above object, the present invention uses following technical proposals：It is multiple based on platinum-nickel alloys load manganese dioxide nano
The preparation of the acetylcholinesteraseelectrochemistry electrochemistry biosensor of condensation material, it includes the following steps：First, with KMnO4、CTAB、
PEG400 and sodium citrate, sodium borohydride, PdCl2And H2PtCl6For starting material, Pd-Pt@MnO are prepared using water bath Method2
Nanocomposite.Using drop-coating by the Pd-Pt@MnO of preparation2Modify glassy carbon electrode surface；Secondly, with chitosan (CS)
Fixed acetylcholinesterase (AChE), Pd-Pt@MnO are fixed on using physical absorption by acetylcholinesterase (AChE)2Modify glass
Carbon electrodes are based on Pd-Pt@MnO2Good chemical property is built into electrochemica biological sensor and is applied to methyl
Quick, the Sensitive Detection of the pesticides such as parathion and Furadan, building process and detection mechanism：
Acetylcholine ester enzymatic substrate acetylthiocholine hydrolyzes, and generates product thiocholine and acetic acid.Thiocholine
It is easily aoxidized under specific potential, generates electric current.Under conditions of concentration of substrate is certain, current signal size and biosensor
Catalytic response it is related.In the case where there is pesticide existence condition, hydroxy combining in the activity of pesticide and acetylcholinesterase is generated to sulphur
For the phosphorylated acetylcholinesterase of acetylcholine catalytically inactive：
Acetylcholinesterase is inhibited the catalytic activity of substrate by pesticide, and the thiocholine concentration of generation declines, electricity
Stream reduces increases of the with pesticide concentration, increases the inhibiting rate of enzyme electrode, calculates percent inhibition [15] according to the following formula:
Inhibiting rate=× 100% (1) [(I0-I1)/I0]
(1) in formula, I0 refers to the stable state electricity that acetylcholine ester enzyme sensor generates certain density acetylthiocholine
Stream, refers to the steady-state current that acetylcholine ester enzyme sensor generates same concentration acetylthiocholine after pesticide inhibits, and is led to
It crosses the current signal compared before and after enzyme inhibits and aoxidizes peak size, realize to parathion-methyl, the agricultures such as sevin and Furadan
The quantitative detection of medicine.
The acetylcholinesterasebiosensor biosensor of the present invention is obtained via following steps：
(1)Pd-Pt@MnO2The preparation of-nanoflakes nanocomposites
①MnO2The preparation of-nanoflakes materials
911mg cetyl trimethylammonium bromides are dissolved in the ultra-pure water of 25ml, are heated to reflux to temperature and are maintained at 140 DEG C
When, it is subsequently added into KMnO4Its quality is that 79.02mg is dissolved in the ultra-pure water of 25mL, is flowed back when temperature is constantly maintained at 140 DEG C
8 to 10 minutes, color became dark-brown from purple, was cooled to room temperature, with ethyl alcohol wash three times, with water be settled to 10mL to get to
MnO2- nanoflakes nanocomposites；
②Pd-Pt@MnO2The preparation of-nanoflakes composite materials
Take 5mL 1. MnO obtained20.5mL 10mM PdCl are added in nanoflakes materials2, 0.5mL10mM
H2PtCl6, 0.1mL PEG400 and 1.0mL 10mM sodium citrates stir and evenly mix, and are eventually adding the reaction of 3mL15mM sodium borohydrides
4h is washed with water centrifuges three times later, is settled to 5mL to get to Pd-Pt@MnO2Composite material；
(2) glassy carbon electrode surface pre-processes
By the glass-carbon electrode Glassy carbon electrode Al of a diameter of 3mm2O3Polishing treatment is pasted into minute surface, according to
Secondary ethyl alcohol and redistilled water are cleaned by ultrasonic, and are positioned in air and spontaneously dry, then in the PBS solution of pH 7.4 in+
300s is scanned under 1.75V constant potentials, scan round 20 times, wait for electric current between+0.3V~+1.25V and+0.3V~-1.3V
After stabilization, rinses electrode with secondary water and spontaneously dry in air spare；
(3) structure at electrochemical biosensor interface
Take the Pd-Pt@MnO that 6uL is obtained2Hanging drop is applied to the glassy carbon electrode surface anticipated, and is placed at room temperature
It is dry to get to Pd-Pt@MnO2Modified glassy carbon electrode is denoted as Pd-Pt@MnO2/GCE；Finally wait for Pd-Pt@MnO2 modification glass carbon
Followed by 4.5 μ L acetylcholinesterases (AChE) of electrode surface drop coating 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 to get to AChE-CS/Pd-Pt@MnO2/
GCE；
(4) standard curve to parathion-methyl and Furadan Pesticides Testing is established
AChE-CS/Pd-Pt@MnO2/ GCE is as working electrode, saturated calomel electrode (saturated calomel
Electrode reference electrode, platinum electrode (platinum wire electrode)) is used as to be used as to electrode, it will when measuring pesticide
Working electrode is placed in the middle inhibition 4min cyclic voltammetry scans containing various concentration parathion-methyl and various concentration Furadan
20 times, after background current stabilization, 10min is incubated at 25 DEG C in mercaptoacetyl choline (ATCl) solution of 2mmol/L, is recorded
Cyclic voltammogram caused by each, current value draw standard curve simultaneously with the variation that the various concentration of pesticide is in rule
The optimum linear range and detection for determining pesticide limit；The range of linearity of parathion-methyl Pesticides Testing is (10-13) mol/L~
(10-8) mol/L, it detects and 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, it detects and 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 are：
1, Pd-Pt@MnO are prepared using the method for water-bath2Nanocomposite, with excellent electro catalytic activity and life
Object compatibility；
2, constructed electrochemica biological sensor is successfully realized to the highly sensitive of parathion-methyl and Furadan pesticide
Detection, the wherein range of linearity of parathion-methyl Pesticides Testing are (10-13) mol/L~(10-8) mol/L, it detects and 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, detection limit
It is (0.3 × 10-12)mol/L；
3, the present invention is based on pesticides to the inhibiting effect of enzyme modified electrode, realizes the mesh of Electrochemical Detection pesticide residue
, sample needed for this method is few, detection time is short and sensitivity is higher, is suitable for the analysis and detection of pesticide residue.
Description of the drawings
Fig. 1 is the cyclic voltammogram of several different materials electrodes, an irreversible oxidation peak all occurs 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@MnO2It is different that/AChE-CS/GCE is applied to detection
The DPV of concentration parathion-methyl schemes.
The line of Fig. 3 black is electrochemica biological sensor electrode Pd-Pt@MnO2It is different that/AChE-CS/GCE is applied to detection
The i-t curves of concentration Furadan pesticide.
The line of Fig. 4 black is electrochemica biological sensor electrode Pd-Pt@MnO2It is different that/AChE-CS/GCE is applied to detection
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 detection various concentration
The standard curve of Furadan pesticide.
Specific implementation mode
In embodiment acetylcholinesterasebiosensor biosensor prepare via step with described in Summary.
Embodiment one：
First, the Pd-Pt@MnO of good dispersion are prepared2Nanocomposite：911mg cetyl trimethylammonium bromides are molten
In the ultra-pure water of 25mL, it is heated to reflux when being maintained at 140 DEG C, is subsequently added into KMnO4Its quality is that 79.02mg is dissolved in 25mL's
Ultra-pure water flows back 8 to 10 minutes when temperature is constantly maintained at 140 DEG C, and color becomes dark-brown from purple, and cooling room temperature is used
Absolute ethyl alcohol is washed three times, and 10mL (being equivalent to 6mg/mL) is settled to get to MnO with water2- nanoflakes materials.Take 5mL systems
The MnO obtained20.5mL 10mM PdCl are added in-nanoflakes materials2, 0.5mL 10mM H2PtCl6, 0.1mLPEG400 and
1.0mL10mM sodium citrates stir and evenly mix, and are eventually adding 3mL 15mM sodium borohydrides reaction 4h, are washed with water later three times, centrifugation,
5mL is settled to get to Pd-Pt@MnO2Composite material.
Take 6uL Pd-Pt@MnO obtained above2Hanging drop is applied to the surfaces glass-carbon electrode GCE anticipated, and is placed on
It is dried at room temperature to get to Pd-Pt@MnO2Modified glassy carbon electrode is denoted as Pd-Pt@MnO2/GCE；Finally in the Pd-Pt@done
MnO2The 4.5 μ L acetylcholinesterases (AChE) of glassy carbon electrode surface drop coating and chitosan (CS) mixture of modification, in 4 DEG C of environment
In be dried overnight to get to AChE-CS/Pd-Pt@MnO2/GCE；
Select 10-13Mol/L~10-8The parathion-methyl pesticide of mol/L concentration ranges is at PBS (0.1mol/L pH 7.4)
It is middle to use cyclic voltammetry scan 20 times, after background current stabilization, in the mercaptoacetyl choline ATCl solution of 2mmol/L at 25 DEG C
Warm 10min is educated, cyclic voltammogram (as shown in Figure 2) caused by each is recorded.It is quantitative determined (such as Fig. 2 institutes with DPV
Show), standard curve (as shown in Figure 4) is established, determines that the detection of the electrochemica biological sensor is limited to 0.35 × 10-13mol/L。
Embodiment two：
First, the Pd-Pt@MnO of good dispersion are prepared2Nanocomposite：911mg cetyl trimethylammonium bromides are molten
In the ultra-pure water of 25mL, it is heated to reflux when being maintained at 140 DEG C to temperature, is subsequently added into KMnO4Its quality is dissolved in for 79.02mg
140 DEG C in the ultra-pure water of 25mL, flow back 8 to 10 minutes when temperature is constantly maintained at 140 DEG C, color becomes dark brown from purple
Color, cooling room temperature are washed three times with absolute ethyl alcohol, and 10mL (being equivalent to 7mg/mL) is settled to get to Pd-Pt MnO with water2It is multiple
Condensation material.
Take the Pd-Pt@MnO that 6uL is obtained2Hanging drop is applied to the surfaces glass-carbon electrode GCE anticipated, and is placed on room temperature
Lower drying is to get to Pd-Pt@MnO2Modified glassy carbon electrode is denoted as Pd-Pt@MnO2/GCE；Finally in the Pd-Pt@MnO done2
The 4.5 μ L acetylcholinesterases (AChE) of glassy carbon electrode surface drop coating and chitosan (CS) mixture of modification are done in 4 DEG C of environment
It is dry, overnight to get to AChE-CS/Pd-Pt@MnO2/GCE；
Select 10-12Mol/L~10-8The Furadan pesticide of mol/L concentration ranges is used in PBS (0.1mol/L pH7.4)
Cyclic voltammetry scan 20 times educates temperature in the mercaptoacetyl choline ATCl solution of 2mmol/L after background current stabilization at 25 DEG C
10min records cyclic voltammogram (as shown in Figure 1) caused by each.It is quantitative determined (such as Fig. 3 institutes with i-t curves
Show), standard curve (as shown in Figure 5) is established, determines that the detection of the electrochemica biological sensor is limited to 0.3 × 10-12mol/L。
Claims (2)
1. a kind of acetylcholinesterasebiosensor biosensor, it is characterised in that the acetylcholinesterasebiosensor biosensor is via following step
Suddenly it obtains：
（1）Pd-Pt@ MnO2The preparation of nanocomposite
①MnO2The preparation of nano material
911mg cetyl trimethylammonium bromides are dissolved in the ultra-pure water of 25ml, are heated to reflux when being maintained at 140 DEG C to temperature,
It is subsequently added into KMnO4Its quality is that 79.02mg is dissolved in the ultra-pure water of 25mL, flows back 8 when temperature is constantly maintained at 140 DEG C
To 10 minutes, color became dark-brown from purple, was cooled to room temperature, with ethyl alcohol wash three times, with water be settled to 10mL to get to
MnO2Nanocomposite；
②Pd-Pt@MnO2The preparation of nanocomposite
Take 5mL 1. MnO obtained20.5mL 10mM PdCl are added in nano material2, 0.5mL 10mM H2PtCl6 , 0.1mL
Polyethylene glycol 400 and 1.0mL 10mM sodium citrates stir and evenly mix, and are eventually adding the reaction of 3mL 15mM sodium borohydrides 4h, Zhi Houyong
Washing centrifuges three times, is settled to 5mL to get to Pd-Pt@MnO2Nanocomposite；
（2）Glassy carbon electrode surface pre-processes
By the glass-carbon electrode Al of a diameter of 3 mm2O3Polishing treatment is pasted into minute surface, uses ethyl alcohol and redistilled water ultrasound clear successively
It washes, is positioned in air and spontaneously dries, then scan 300s under+1. 75 V constant potentials in the PBS solution of pH 7. 4,
Scan round is multiple between 3 V of+0. 3 V~+1. 25 V and+0. 3 V~-1., waits for that electric current is stablized
Afterwards, it rinses electrode with secondary water and spontaneously dries in air spare；
（3）The structure at electrochemical biosensor interface
Take the Pd-Pt@MnO that 6 μ L are obtained2Hanging drop is applied to the glassy carbon electrode surface anticipated, and puts and is dried at room temperature for,
Obtain Pd-Pt MnO2Modified glassy carbon electrode is denoted as Pd-Pt@MnO2/GCE；Finally wait for that Pd-Pt@MnO2 modified glassy carbon electrodes are dry
Afterwards followed by 4. 5 μ L acetylcholinesterases of electrode surface drop coating and chitosan compound, wherein：Acetylcholinesterase is 3
μ L and chitosan are 1.5 μ L, are dried overnight under 4 DEG C of environment to get to acetylcholinesterase-chitosan/Pd-Pt@MnO2/
GCE；
（4）Establish the standard curve to parathion-methyl and Furadan Pesticides Testing
Acetylcholinesterase-chitosan/Pd-Pt@MnO2/ GCE as working electrode, saturated calomel electrode as reference electrode,
Platinum electrode is used as to electrode, and working electrode is placed in containing various concentration parathion-methyl and various concentration furans when measuring pesticide
Inhibit 4min cyclic voltammetry scans multiple in red test solution, after background current stabilization, in the mercaptoacetyl choline of 2mmol/L
Incubate 10 min in solution at 25 DEG C, record cyclic voltammogram caused by each, current value with pesticide various concentration
In the variation of rule, draws standard curve and determine that the optimum linear range of pesticide and detection limit；Parathion-methyl Pesticides Testing
The range of linearity be（10-13）Mol/L~（10-8）Mol/L, detection are limited to（0.35×10-13）mol/L；Furadan pesticide is examined
The range of linearity of survey is（10-12）Mol/L~（10-8）Mol/L, detection are limited to（0.3×10-12）mol/L.
2. acetylcholinesterasebiosensor biosensor described in claim 1 is in parathion-methyl and Furadan Pesticides Testing
Using.
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| CN108120761B (en) * | 2017-11-30 | 2020-10-16 | 宁波大学 | Electrochemical biosensors based on peptidomimetics with electrocatalytic activity for acetylcholinesterase detection |
| CN108776163B (en) * | 2018-06-29 | 2020-10-30 | 武汉中科志康生物科技有限公司 | Enzyme biosensor for detecting cholesterol and preparation method and application thereof |
| CN110031521B (en) * | 2019-05-15 | 2021-07-27 | 贵州大学 | Preparation of acetylcholinesterase biosensor and application of acetylcholinesterase biosensor in detection of organophosphorus |
| CN110186910B (en) * | 2019-05-15 | 2021-11-12 | 济南大学 | Double-inhibition electrochemiluminescence sensor and method for measuring glyphosate |
| CN110320187B (en) * | 2019-06-20 | 2021-08-24 | 东南大学 | Detection of organophosphorus pesticide by ratio-type fluorescent sensor based on manganese dioxide nanosheet |
| CN111189822B (en) * | 2020-02-17 | 2021-06-22 | 吉林大学 | Target response type hydrogel and smart phone integrated organophosphorus pesticide field quantitative detection platform |
| CN111521658A (en) * | 2020-06-12 | 2020-08-11 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method of high-sensitivity sensor for pesticide detection, product and application thereof |
| CN112114016B (en) * | 2020-09-22 | 2022-09-23 | 广西壮族自治区农业科学院农产品质量安全与检测技术研究所 | Electrochemical method for detecting 3-nitropropionic acid |
| CN112378975B (en) * | 2020-11-12 | 2022-07-05 | 济南大学 | Electrochemical sensor for detecting AChE inhibitor |
| CN114134138B (en) * | 2021-12-13 | 2023-06-16 | 辽宁大学 | Ionic liquid polymer-based electrochemical modification material for pesticide detection and preparation method and application thereof |
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