CN107490609B - Acetamiprid aptamers electrochemical sensor based on mesoporous silicon dioxde film - Google Patents
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Abstract
A kind of preparation method of electrochemical aptamer sensor and its application in Acetamiprid detection, by the integration of aptamers door control system and the vertical orderly mesoporous silicon dioxde film (MSF) for being grown in the surface indium-tin oxide electrode (ITO), a simple, sensitive electrochemical aptamer sensor platform is constructed.On this aptamer sensor platform, aptamers can be effectively adsorbed in the amidized surface MSF by non-covalent electrostatic attraction, be used as ideal grid material, to control the closing and release of the electrochemistry reagent methylene blue captured in the hole MSF.In the presence of Acetamiprid, the specific binding of aptamers and Acetamiprid can trigger the unlatching in hole, release methylene blue, reduce the current signal of detection.This electrochemical aptamer sensor design is simple, easy to operate, can be realized quick, the sensitive detection to Acetamiprid.
Description
Technical field
The present invention relates to pesticide residue fields, are adapted in particular to a kind of electrochemistry based on MSFs modified electrode
The preparation of body sensor and the detection of Acetamiprid.
Background technique
Pesticide generally has immunotoxicity, neurotoxicity, genetic toxicity and three causing toxicity etc., in order to improve and ensure
Food quality and life security are increasingly valued by people the highly sensitive detection of pesticide residue.Pesticide residue is examined at present
The method of survey mainly has gas-chromatography, high performance liquid chromatography, Chromatography/Mass Spectrometry joint technology, Capillary Electrophoresis, surface-enhanced Raman
Spectroscopic methodology, immunoassay, biosensor etc..Gas-chromatography, high performance liquid chromatography and Chromatography/Mass Spectrometry joint technology, separation effect
Can high, high sensitivity, but expensive equipment and be not easy to realize micromation, the pretreatment process of sample is relatively complicated；Capillary
Electrophoresis then has many advantages, such as that clastotype is more, high-efficient, analysis speed is fast, reagent and sample consumption are few, but capillary diameter is small,
Optical path is short, poor reproducibility；Surface enhanced Raman spectroscopy method has very high sensitivity, but reproducibility and stability are poor.Electrochemistry
Sensor is with its low-power consumption, high sensitivity, high-precision, strong antijamming capability, the range of linearity are wide and excellent repeatability, stability
Etc. characteristics, be widely applied to the every field such as biology, environment, food.
Summary of the invention
The purpose of the present invention is provide one kind for the disadvantage in above-mentioned Detecting Pesticide with aptamers door control system
With the integration for the vertical orderly mesoporous silicon dioxde film (MSF) for being grown in the surface indium-tin oxide electrode (ITO), an electricity is constructed
Chemical aptamer sensor platform, for quick, the Sensitive Detection to Acetamiprid.
The technical solution of the present invention is as follows: a kind of electrochemical method for detecting Acetamiprid pesticide, specific steps are as follows: graphite oxide
The preparation of alkene: weighing 6.8 g potassium permanganate, is added slowly with stirring the 120 mL concentrated sulfuric acids and 14 mL phosphoric acid；1.0 g are added
Graphite powder keeps reaction temperature to be lower than 20 DEG C, stirs 15 min, later adds to mixed liquor in three-necked flask dropwise, at 50 DEG C
Continuous uniform stirs 12 h in water-bath；140 mL ice water are added and are allowed to cooling, are slowly added to the hydrogen peroxide of 3.0 mL 30%, at this time
Solution is in glassy yellow, and 2 h are ultrasonically treated after being cooled to room temperature, and is then centrifuged with the revolving speed of 6000 r/s, supernatant is removed, with steaming
Distilled water washing is precipitated to neutrality, is placed at 60 DEG C dry 12 h, obtains graphene oxide；
The preparation of gold-poly- 3,4- ethene dioxythiophene/redox graphene (Au-PEDOT/rGO) nanocomposite:
The ethanol solution of 5 mL 3,4- ethene dioxythiophenes (EDOT) (22.5 mmol/L) is poured into 70 mL while stirring at room temperature
HAuCl4In aqueous solution (0.65 mmol/L), mixture is immediately turned into navy blue.In this process, EDOT(3,4- ethylene dioxy
Thiophene) monomer is oxidized and polymerize to obtain PEDOT, and HAuCl4Ion is reduced as oxidant, is formed simultaneously AuNP.It will
Reaction is kept stirring 4 hours, and 5 mL GO aqueous dispersions (0.5 mg/mL) are then added, obtains mixture solution and (is expressed as Au-
PEDOT/GO)；The Au-PEDOT/GO of acquisition is ultrasonically treated 2 hours, keeps Au-PEDOT and GO fully dispersed；Then, quick
By 5.5 mL NaBH under stirring4Aqueous solution (0.16 mol/L) is slowly dropped in mixture, and reaction system is kept stirring
6 hours；Finally, the mixture (Au-PEDOT/rGO) of acquisition is centrifuged and is washed repeatedly with water and ethyl alcohol, then by solid point
It is dispersed in 50 mL deionized waters for future use；
The preparation of electrochemical aptamer sensor: ITO electrode is cleaned by ultrasonic 15 in acetone, second alcohol and water respectively first
Min, by 10 μ L Au-PEDOT/rGO drop coatings in clean ITO electrode surface, and it is dry in 45 DEG C of baking oven；It will modification
The ITO electrode of base material immerse containing 35 mL water, 15 mL ethyl alcohol, 0.08 g CTAB, 5 μ L ammonia spirits (25%) and
In the mixture solution of 40 μ L TEOS, MSFs is allowed to grow 48 hours under 60 DEG C, quiescent conditions, by ITO electrode ethyl alcohol and
Water rinsing, and be dried overnight at 60 DEG C；The modified electrode of MSFs is immersed into the ethyl alcohol containing 0.1 mol/L HCl under stiring
In 5 minutes, to remove ctab surface activating agent.Finally, MSFs modified ITO electrode is immersed the second that 4 mL contain 5%APTES
12 hours in alcoholic solution, gently shakes, obtain NH2- MSFs, being dipped in methylene blue concentration is 10-5The Tris- of mol/L
In HCl solution (10 mmol/L, pH=7.4), and shaken overnight at room temperature；Then, by 10 μ L 10-8The adaptation of mol/L
Liquid solution is added drop-wise to the MSFs modified electrode surface of load methylene blue, is incubated at room temperature 2 hours, obtains aptamers sealing end
MSFs modified electrode；After being incubated for aptamers, by 10 mmol/L Tris-HCl buffers (pH=7.4) of modified ITO electrode
Washing is to remove non-specific adsorption；At room temperature, the 10 μ L of MSFs modified electrode immersion of aptamers sealing end are contained a certain amount of
2 hours in the buffer solution of Acetamiprid；Finally, being removed by thoroughly being washed with buffer suitable with Acetamiprid specific binding
Ligand.
A kind of electrochemical method detecting Acetamiprid, specific rapid as follows:
It (1) is working electrode by the ITO of above-mentioned modification in the electrolytic cell of the PBS solution containing 10 mL pH 7.0,
Ag/AgCl electrode is reference electrode, and platinum electrode is to electrode；Experiment carries out on CHI842C Electrochemical Comprehensive Tester, attached
The computer software of category is made for the acquisition and processing of experimental data；Differential pulse is carried out in -0.5~0.1 V potential range to sweep
It retouches, records differential pulse voltammetry voltammogram, peak point current when Acetamiprid is not added in measurementIp 0 ；According to said method, the pyridine of various concentration is added
Corresponding peak point current can be obtained in worm amidineIp x , calculate peak current difference △Ip(△Ip=I p0 - I px )；△Ip/Ip 0 With pyridine
The logarithm of worm amidine concentrationlgcGood linear relationship, linear equation △ is presentedIp/Ip 0 =0.49258+0.05731 lgc, c
It is concentration, unit is mol/L, △IpIt is peak current difference, unit is mA, linearly dependent coefficient R=0.99477, Acetamiprid concentration
The range of linearity be 10-8 mol/L~10-13Mol/L, sensor are limited to 3.4 × 10 to the detection of Acetamiprid pesticide-14mol/L；
(2) above-mentioned linear relationship is combined, the Acetamiprid sample of unknown concentration is measured, Acetamiprid concentration is calculated:
In the electrolytic cell of the PBS solution containing 10 mL pH 7.0, using the ITO of above-mentioned modification as working electrode, Ag/AgCl electrode
For reference electrode, platinum electrode is to electrode；Experiment carries out on CHI842C Electrochemical Comprehensive Tester, attached computer
Software is made for the acquisition and processing of experimental data；A certain amount of solution to be measured is added, is carried out in -0.5~0.1 V potential range
Differential pulse scanning, records differential pulse voltammetry voltammogram, obtains peak point currentI p, willI pAbove-mentioned equation is substituted into, calculating prepare liquid can be sought
The concentration of middle Acetamiprid.
The beneficial effects of the present invention are: the present invention by aptamers door control system be grown in the vertical of ITO electrode surface
The ingenious integration of orderly MSFs is prepared for a simple, light, unmarked electrochemical aptamer sensor platform；Use adaptation
Body as grid material, come control MSFs mesoporous Methylene Blue obstruction and release；Based between aptamers and target substance
High-affinity and specificity, aptamers the surface MSFs is only adsorbed on by non-covalent electrostatic attraction, this method is easy to operate
And without the chemical modification of any complexity；Prepared electrochemical aptamer sensor shows Acetamiprid highly sensitive and good
Good selectivity, the experimental results showed that, the electrochemical aptamer sensor proposed has in trace Pesticides Testing and potentially answers
Use prospect.
Detailed description of the invention:
DPV signal strength stacking chart under Fig. 1 difference Acetamiprid concentration
Wherein, 1-0 mol/L, 2-10-13Mol/L, 3-10-12Mol/L, 4-10-11Mol/L,
5—10-10Mol/L, 6-10-9Mol/L, 7-10-9mol/L；
Ratio and lg of Fig. 2 peak current difference of the present invention with current value when being not added AcetamipridcLinear relationship chart.
Specific embodiment:
For a better understanding of the present invention, below with specific example come the technical solution that the present invention will be described in detail, but this
Invention is not limited thereto.
Embodiment 1
1. the preparation of graphene oxide
6.8 g potassium permanganate are weighed, the 120 mL concentrated sulfuric acids and 14 mL phosphoric acid are added slowly with stirring；1.0 g stones are added
Ink powder keeps reaction temperature to be lower than 20 DEG C, stirs 15 min, later adds to mixed liquor in three-necked flask dropwise, in 50 DEG C of water
Continuous uniform stirs 12 h in bath；140 mL ice water are added and are allowed to cooling, are slowly added to the hydrogen peroxide of 3.0 mL 30%, it is molten at this time
Liquid is in glassy yellow, and 2 h are ultrasonically treated after being cooled to room temperature, and is then centrifuged with the revolving speed of 6000 r/s, supernatant is removed, with distillation
Water washing is precipitated to neutrality, is placed at 60 DEG C dry 12 h, obtains graphene oxide；
2. the preparation of Au-PEDOT/rGO nanocomposite
The preparation of gold-poly- 3,4- ethene dioxythiophene/redox graphene (Au-PEDOT/rGO) nanocomposite:
The ethanol solution of 5 mL 3,4- ethene dioxythiophenes (EDOT) (22.5 mmol/L) is poured into 70 mL while stirring at room temperature
HAuCl4In aqueous solution (0.65 mmol/L), mixture is immediately turned into navy blue.In this process, EDOT(3,4- ethylene dioxy
Thiophene) monomer is oxidized and polymerize to obtain PEDOT, and HAuCl4Ion is reduced as oxidant, is formed simultaneously AuNP.It will
Reaction is kept stirring 4 hours, and 5 mL GO aqueous dispersions (0.5 mg/mL) are then added, obtains mixture solution and (is expressed as Au-
PEDOT/GO)；The Au-PEDOT/GO of acquisition is ultrasonically treated 2 hours, keeps Au-PEDOT and GO fully dispersed；Then, quick
By 5.5 mL NaBH under stirring4Aqueous solution (0.16 mol/L) is slowly dropped in mixture, and reaction system is kept stirring
6 hours；Finally, the mixture (Au-PEDOT/rGO) of acquisition is centrifuged and is washed repeatedly with water and ethyl alcohol, then by solid point
It is dispersed in 50 mL deionized waters for future use；
3. the preparation of electrochemical aptamer sensor
ITO electrode is cleaned by ultrasonic 15 min in acetone, second alcohol and water respectively first, by 10 μ L Au-PEDOT/rGO
Drop coating is dry in clean ITO electrode surface, and in 45 DEG C of baking oven；The ITO electrode for having modified base material is immersed
Containing 35 mL water, 15 mL ethyl alcohol, 0.08 g CTAB, 5 μ L ammonia spirits (25%) and 40 μ L TEOS mixture solution
In, it allows MSFs to grow 48 hours under 60 DEG C, quiescent conditions, ITO electrode second alcohol and water is rinsed, and dried at 60 DEG C
Night；The modified electrode of MSFs is immersed in the ethyl alcohol containing 0.1 mol/L HCl 5 minutes under stiring, to remove ctab surface
Activating agent.Contain in the ethanol solution of 5%APTES 12 hours finally, MSFs modified ITO electrode is immersed 4 mL, gently shakes
It is dynamic, obtain NH2- MSFs, being dipped in methylene blue concentration is 10-5Mol/L Tris-HCl solution (10 mmol/L, pH=
7.4) in, and shaken overnight at room temperature；Then, by 10 μ L 10-8The adaptation liquid solution of mol/L is added drop-wise to load methylene
Blue MSFs modified electrode surface, is incubated at room temperature 2 hours, obtains the MSFs modified electrode of aptamers sealing end；Use aptamers
After incubation, modified ITO electrode is washed with 10 mmol/L Tris-HCl buffers (pH=7.4) to remove non-specific suction
It is attached；At room temperature, the MSFs modified electrode of aptamers sealing end 10 μ L are immersed to contain 2 in the buffer solution of a certain amount of Acetamiprid
Hour；Finally, removing the aptamers with Acetamiprid specific binding by thoroughly being washed with buffer.
4. utilizing the content of above-mentioned aptamer sensor detection Acetamiprid
It (1) is working electrode by the ITO of above-mentioned modification in the electrolytic cell of the PBS solution containing 10 mL pH 7.0,
Ag/AgCl electrode is reference electrode, and platinum electrode is to electrode；Experiment carries out on CHI842C Electrochemical Comprehensive Tester, attached
The computer software of category is made for the acquisition and processing of experimental data；Differential pulse is carried out in -0.5~0.1 V potential range to sweep
It retouches, records differential pulse voltammetry voltammogram, peak point current when Acetamiprid is not added in measurementIp 0 ；According to said method, the pyridine of various concentration is added
Corresponding peak point current can be obtained in worm amidineIp x , calculate peak current difference △Ip(△Ip=I p0 - I px )；△IpWith Acetamiprid
The logarithm of concentrationlgcGood linear relationship, linear equation △ is presentedIp/Ip 0 =0.49258+0.05731 lgc, c are dense
Degree, unit is mol/L, △IpIt is peak current difference, unit is mA, linearly dependent coefficient R=0.99477, the line of Acetamiprid concentration
Property range be 10-8 mol/L~10-13Mol/L, sensor are limited to 3.4 × 10 to the detection of Acetamiprid pesticide-14mol/L；
(2) above-mentioned linear relationship is combined, the Acetamiprid sample of unknown concentration is measured, Acetamiprid concentration is calculated:
In the electrolytic cell of the PBS solution containing 10 mL pH 7.0, using the ITO of above-mentioned modification as working electrode, Ag/AgCl electrode
For reference electrode, platinum electrode is to electrode；Experiment carries out on CHI842C Electrochemical Comprehensive Tester, attached computer
Software is made for the acquisition and processing of experimental data；A certain amount of solution to be measured is added, is carried out in -0.5~0.1 V potential range
Differential pulse scanning, records differential pulse voltammetry voltammogram, obtains peak point currentI p, willI pAbove-mentioned equation is substituted into, calculating prepare liquid can be sought
The concentration of middle Acetamiprid.
Claims (2)
1. a kind of preparation method for the electrochemical aptamer sensor for detecting Acetamiprid, which is characterized in that specific steps are as follows: will obtain
The mixture Au-PEDOT/rGO obtained is centrifuged and is washed repeatedly with water and ethyl alcohol, and then solid is dispersed in 50mL deionized water
For future use；It is finally the preparation of electrochemical aptamer sensor, it is first that ITO electrode is ultrasonic in acetone, second alcohol and water respectively
Clean 15min；By 10 μ L Au-PEDOT/rGO ink drop in clean ITO electrode surface, and done in 45 DEG C of baking oven
It is dry；The ITO electrode for having modified base material is immersed molten containing 35mL water, 15mL ethyl alcohol, 0.08g CTAB, 5 μ L, 25% ammonium hydroxide
In the mixture solution of liquid and 40 μ L TEOS, MSFs is allowed to grow 48 hours under 60 DEG C, quiescent conditions；It, will after MSFs growth
ITO electrode is rinsed with second alcohol and water, and is dried overnight at 60 DEG C；The modified electrode of MSFs is immersed under stiring and is contained
5 minutes in the ethyl alcohol of 0.1mol/L HCl, to remove ctab surface activating agent；Finally, MSFs modified ITO electrode is immersed
4mL contains in the ethanol solution of 5%APTES 12 hours, gently shakes, to ensure that the surface of MSFs can further use amino official
Energyization；After MSFs modified ITO electrode amination, immersing methylene blue concentration is 10-5The 10mmol/L pH7.4's of mol/L
In Tris-HCl solution, and shaken overnight at room temperature；Then, by 10 μ L 10-8The adaptation liquid solution of mol/L is added drop-wise to load
The MSFs modified electrode surface of methylene blue, is incubated at room temperature 2 hours, obtains the MSFs modified electrode of aptamers sealing end；With
After aptamers are incubated for, modified ITO electrode is washed with the Tris-HCl buffer of 10mmol/L pH7.4 non-specific to remove
Property absorption；At room temperature, the MSFs modified electrode of aptamers sealing end is immersed into the buffer solution that 10 μ L contain a certain amount of Acetamiprid
In 2 hours；Finally, removing the aptamers with Acetamiprid specific binding by thoroughly being washed with buffer.
2. electrochemical aptamer prepared by the preparation method of electrochemical aptamer sensor according to claim 1 senses
Application of the device in detection Acetamiprid, which is characterized in that specific steps are as follows:
It (1) is working electrode, Ag/AgCl by the ITO of above-mentioned modification in the electrolytic cell of the PBS solution containing 10mL pH 7.0
Electrode is reference electrode, and platinum electrode is to electrode；Experiment carries out on CHI842C Electrochemical Comprehensive Tester, attached meter
Calculation machine software is made for the acquisition and processing of experimental data；Differential pulse scanning, record are carried out in -0.5~0.1V potential range
Peak point current Ip when Acetamiprid is not added in differential pulse voltammetry voltammogram, measurement0；According to said method, the Acetamiprid of various concentration is added, i.e.,
Corresponding peak point current Ip can be obtainedx, calculate peak current difference DELTA Ip, Δ Ip=Ip0-Ipx；Pair of Δ Ip and Acetamiprid concentration
Good linear relationship is presented in number lgc, and linear equation is Δ Ip/Ip0=0.49258+0.05731lgc, c are concentration, and unit is
Mol/L, Δ Ip are peak current differences, and unit is μ A, and the range of linearity of linearly dependent coefficient R=0.99477, Acetamiprid concentration are
10-8Mol/L~10-13Mol/L, sensor are limited to 3.4 × 10 to the detection of Acetamiprid pesticide-14mol/L；
(2) above-mentioned linear relationship is combined, the Acetamiprid sample of unknown concentration is measured, Acetamiprid concentration is calculated: being contained
In the electrolytic cell for having the PBS solution of 10mL pH 7.0, using the ITO of above-mentioned modification as working electrode, Ag/AgCl electrode is reference
Electrode, platinum electrode are to electrode；Experiment carries out on CHI842C Electrochemical Comprehensive Tester, and attached computer software supplies
Make the acquisition and processing of experimental data；A certain amount of solution to be measured is added, differential pulse is carried out in -0.5~0.1V potential range
Scanning records differential pulse voltammetry voltammogram, obtains peak point current Ip, Ip is substituted into above-mentioned equation, can ask and calculate pyridine worm in prepare liquid
The concentration of amidine.
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| CN110455897B (en) * | 2019-08-29 | 2021-07-02 | 济南大学 | Based on SiO2Sensitive detection of Hg by carrier2+Construction of the Release electrochemical aptamer sensor |
| CN114624298B (en) * | 2020-12-12 | 2023-05-05 | 中国科学院大连化学物理研究所 | Method for detecting ochratoxin A by using electrochemical paper chip based on aptamer gating effect |
| CN113447553B (en) * | 2021-06-21 | 2022-09-20 | 同济大学 | Non-immobilized electrochemical sensor based on signal probe packaging release and application thereof |
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| CN106706737A (en) * | 2016-12-10 | 2017-05-24 | 武汉市农业科学技术研究院农业环境安全检测研究所（武汉市农业科学技术研究院中心实验室） | Rapid detection method for ochratoxin A |
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| CN104897746A (en) * | 2015-05-06 | 2015-09-09 | 同济大学 | Preparation method of aptamer photoelectrochemical sensor for high-sensitivity high-selectivity detection of MC-LR |
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| CN102980935A (en) * | 2012-12-06 | 2013-03-20 | 济南大学 | Electrochemical method for detecting anthracene-phenanthrene resultant of polycyclic aromatic hydrocarbon |
| CN106706737A (en) * | 2016-12-10 | 2017-05-24 | 武汉市农业科学技术研究院农业环境安全检测研究所（武汉市农业科学技术研究院中心实验室） | Rapid detection method for ochratoxin A |
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