CN110455898A - A kind of high-ratio surface nanogold makees the preparation of the electrochemical sensor of signal amplification carrier and its application in combined toxicity of pesticides evaluation - Google Patents
A kind of high-ratio surface nanogold makees the preparation of the electrochemical sensor of signal amplification carrier and its application in combined toxicity of pesticides evaluation Download PDFInfo
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- 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
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- G—PHYSICS
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- 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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- 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
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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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- 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
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
Abstract
The invention belongs to biosensor technique fields, make the preparation of the electrochemical sensor of signal amplification carrier more particularly to a kind of high-ratio surface nanogold and its in the application of combined toxicity of pesticides evaluation, the following steps are included: Au nanometers of seeds of growth, prepare high-specific area nano gold material, the preparation of aptamer sensor and generation electrochemical signals.This invention ensures that the reliability and stability of sensor；Improve the capture efficiency to target cell；Improve electron transfer rate；Higher level, high sensitivity are shown to the electric signal recognition capability of chlopyrifos.
Description
Technical field
The invention belongs to biosensor technique fields, and in particular to a kind of high-ratio surface nanogold makees signal amplification carrier
The preparation of electrochemical sensor and its application evaluated in combined toxicity of pesticides.
Background technique
Organic phosphorus compound (OPs) is a kind of pesticide for being widely used in agricultural, and insecticidal activity with higher is raw in agricultural
It is widely used in production.It is estimated that the whole world controls and kills crop using more than 200 ten thousand tons of this insecticides every year
The insect and pest of Tanaka.Wherein, chlopyrifos is mainly used for the different types of pest of crops field control, including termite, mosquito
Son, roundworm cut worm, corn rhizome worm, flea beetle, fly, fiery ant, louse etc..Chlopyrifos usually by different approach into
Enter environment, such as rainwater is discharged into surface water, spray drift or the soil erosion, therefore is often sent out in air, food and water
Existing chlopyrifos.It has been reported that fish death incident is related with water body Chlorpyrifos, to confirm that water body Chlorpyrifos content is reachable
A few millionths hundred.The toxicity of chlopyrifos (CP) causes many potential hazards to human health.It is poisoned with poison in addition, being contained by sucking
The air of tick, it can enter human body.It eats contaminated food or directly contact chlopyrifos can lead to serious poisoning.According to report
Road, chlopyrifos can be absorbed by the body by respiratory tract, so as to cause occur coughing, wheeze, the respiratory symptoms such as airway inflammation.
Furthermore, it is also possible to other symptoms occur, such as headache, numbness, harmony problem, dizziness, is trembled, cramp, eyesight at nausea
It is fuzzy, heartbeat is excessively slow etc..The CP of high dose may cause twitch, or even death due to cardiovascular failure.
Carbofuran is a kind of carbamate pesticide, is used to kill the elder brother in crops and plant as broad spectrum pesticide
Worm is widely used in agricultural production., acarid and nematode.Card Budweiser furans residual quantity is strong to the mankind in agricultural product and living environment
Health and animal husbandry constitute potential threat.This is because carbofuran can change the oxidation balance and stability of human red blood cells cell membrane.
Due to its toxicity height, the maximum residue limit (MRL) of cereal and oilseeds is set as 0.1 or 0.2 mgkg by China-1, U.S.'s ring
The MRL of its selected agricultural product (including mung bean, banana, coffee and rice) is set as 0.1 mgkg by border protection administration (EPA)-1.Some countries such as the U.S., Canada and European Union have forbidden using carbofuran in veterinary antibiotics, herbal medicine and tealeaves.Its shadow
The Different Organs for ringing human body, such as brain, liver, muscle and heart.Therefore, carbofuran is able to suppress the function of acetylcholinesterase
Can, to effectively prevent neurotransmission.The detection technique of carbofuran is varied, such as chromatography, electrochemical process, aptamer sensing
Device etc..But chromatography and electrochemical process the disadvantages of there are pre-processing requirements complexity, poor repeatability, poor selectivity.In addition, adapter
Sensor usually requires label and indication signal.
The common detection method of these pesticides is to connect mass spectrum (HPLC- by gas-chromatography (GC) or high performance liquid chromatography
MS), this method has the characteristics that stability and sensibility are high, and many countries are still logical to the national standard of Pesticides Testing now
Cross the detection of the large-scale instruments such as GC or HPLC.But by ingredient, high, sample needs complicated pre-treatment, needs professional people for large-scale instrument detection
Member etc. factors, be difficult in many places it is accomplished, so the exploitation of fast and convenient detection method, is still the side of pesticide research
To.
Electrochemica biological sensor is using bioactive substance as sensitive member, and electrode is as signal adapter, by measured object
Chemical signal be converted into electric signal, the intensity according to electric signal detects target concentration.Metal nano material due to
Its excellent electric conductivity, high-specific surface area and excellent catalytic performance, are widely applied in electrochemical sensor.
Summary of the invention
The purpose of the present invention is to solve the above problem, provides a kind of high-ratio surface nanogold and makees signal amplification carrier
Electrochemical sensor poisons slave girl and carbofuran with poison to the joint toxicity of cell for check and evaluation, has highly sensitive, high stability
And ease for use.
Technical solution according to the invention, the high-ratio surface nanogold make the electrochemical sensor of signal amplification carrier
Preparation, includes the following steps,
(1) it weighs hexadecyltrimethylammonium chloride (CTAC) to be added in 1-50mL deionized water, adjusting CTAC concentration is 0.1-
1.0wt% adds the HAuCl of 0.1-5.0mL, 1-30mM4Reaction solution is maintained in 20-50 DEG C of water-bath by aqueous solution；Then
The KBr solution of 5-100 μ L, 0.1-5.0mM is added, stands 10-30min, it is molten that 5-100 μ L, the KI of 0.1-5.0mM is then added
Liquid, then stand 10-30min, after the L-AA solution for continuously adding 100-800 μ L, 5-100mM, stand 30-60min with
Grow Au nanometers of seeds；
(2) by the HAuCl of CTAC and 0.1-5.0mL, 1-30mM4It is added in 1-50mL deionized water and prepares growth solution, adjust
Section CTAC concentration is 0.1-1.0wt%；The l-Glutathione of 0.1-10.0 μ L, 0.1-50mM are added into growth solution, is inverted examination
Pipe oscillation is uniformly mixed, and Au nanometers of seed solutions obtained by 1.0-100.0 μ L step (1) are then added, and oscillation mixes；By solution
It is placed in 20-50 DEG C of water-bath centrifugal enrichment after 30-60min, is washed 1-3 times, obtained high-specific area nano gold material dispersion
In 3-5mL ultrapure water；
(3) glass-carbon electrode is immersed in etching liquid 5-20 minutes, glass-carbon electrode is then polished to mirror-like with alumina slurry
Surface, successively through ultrasonic cleaning glass-carbon electrode 1-3min in water, ethyl alcohol, water, the glass-carbon electrode after cleaning is in 0.1-2.0M
H2SO4Further electrochemical activation in solution, then with ultrapure water and use N2It is dry；
(4) 1-25 μ L, 0.1-10.0 μM of DNA aptamer drop are handled into gained glassy carbon electrode surface, 0-20 DEG C of incubation in step (3)
Overnight, it is then washed with deionized to remove unbonded DNA aptamer；High-specific surface area obtained by 5-10 μ L step (2) is added dropwise
Nanogold material solution, high-specific area nano gold material are fixed on glass-carbon electrode by Au-S key and form aptamer sensor；
(5) by the PBS solution for the HepG-2 cell that aptamer sensor and pH are 6.0-8.0, the 0.05-0.25M containing concentration in 20-50
It DEG C incubates and to form sandwich shape structure to capture HepG-2 cell in 1-5 hour, then washed 1-3 times with PBS, obtained electrochemistry aptamer
Sensor.
Further, the etching liquid in the step (3) uses H2SO4: 30%H2O2=3:1 etching liquid.
Further, gained aptamer sensor is stored in 0-8 DEG C of refrigerator before use in the step (4).
Further, gained electrochemistry aptamer sensor is placed in 0.1-10mL in Electrochemical Detection in the step (5)
The H of quinhydrones and 0.1-10.0mM containing 0.1-10.0mM2O2PBS solution in.
Another aspect provides the electrochemical sensors that a kind of high-ratio surface nanogold makees signal amplification carrier
The application of combined toxicity of pesticides evaluation, for the detection of chlopyrifos, the electrochemical sensor is to be appointed using such as claim 1-3
Electrochemistry aptamer sensor made from one preparation method, comprising the following steps: 5 μ L chlopyrifos standard solution or sample are molten
Drop is added on the surface of the electrochemical sensor, 30-40 DEG C incubation 1-2 hours, washed with PBS and use N2It is dry；It will be electric
Chemical sensor immerses in the phosphate buffered saline solution of pH 7.4,10mM, is measured using differential pulse voltammetry, sets pulse
Amplitude 50Mv, the differential pulse voltammetry curve being recorded between -0.3V and 0.5V.
It is of the invention to additionally provide a kind of high-ratio surface nanogold and make the electrochemical sensor of signal amplification carrier in pesticide
The application of joint toxicity evaluation, for the joint toxicity evaluation of binary organophosphorus pesticide on a cellular level, the electrochemistry is passed
Sensor is using electrochemistry aptamer sensor made from the preparation method as described in claim 1-3 is any, comprising the following steps: is taken
Treated grows to the HepG-2 cell of logarithmic phase, is added thereto with concentration than the chlopyrifos prepared for 1:1 and carbofuran
Mixed solution, the chlopyrifos of various concentration and carbofuran are diluted with acetone using preceding, add cell culture medium and be formulated into institute
Concentration is needed, and keeps the volumetric concentration of acetone to be lower than 0.5%, and using the cell base containing 0.5% volumetric concentration acetone as experiment
Control group；The concentration of chlopyrifos and carbofuran mixed solution is added in adjustment, and the differential pulse voltammetry for testing electrochemical sensor is rung
It answers.
The beneficial effects of the present invention are:
Due to the good biocompatibility of nanogold, can be used as the carrier of fixed dna aptamer, ensure that sensor reliability and
Stability；
Due to the nanogold large specific surface area of synthesis, surface free energy is high, greatly improves the supported quantity of DNA aptamer, improves
To the capture efficiency of target cell；
Gold nanoparticle has good electronics to mediate ability, can promote the electronics in reaction system between electrode surface
Transmitting improves electron transfer rate；
The sandwich shape electrochemical sensing system of aptamer-cell-DNA nano-probe is constructed in electrode surface, signal amplifying power obtains
To being obviously improved, higher level, high sensitivity are shown to the electric signal recognition capability of chlopyrifos.
Specific embodiment
The present invention is further explained in the light of specific embodiments.
Embodiment one
A kind of high-ratio surface nanogold makees the preparation method of the electrochemical sensor of signal amplification carrier, includes the following steps,
(1) it weighs CTAC to be added in 1mL deionized water, adjusting CTAC concentration is 0.5wt%, adds 0.1mL, 15mM
HAuCl4Reaction solution is maintained in 20 DEG C of water-baths by aqueous solution；Then the KBr solution of 5 μ L, 2.5mM is added, stands 10min, with
The KI solution of 5 μ L, 2.5mM are added afterwards, then stands 10min, after the L-AA solution for continuously adding 100 μ L, 55mM, stands
30min is to grow Au nanometers of seeds；
(2) by the HAuCl of CTAC and 0.1mL, 15mM4It is added in 1mL deionized water and prepares growth solution, adjust CTAC concentration
For 0.5wt%；Into growth solution be added 0.1.0 μ L, 25mM l-Glutathione, be inverted test tube oscillation be uniformly mixed, then plus
Enter Au nanometers of seed solutions obtained by 1.0 μ L steps (1), oscillation mixes；Solution is placed in 20 DEG C of water-baths after 30min and is centrifuged richness
Collection washs 1 time, and obtained high-specific area nano gold material is dispersed in 3mL ultrapure water；
(3) glass-carbon electrode is immersed into etching liquid (H2SO4: 30%H2O2=3:1) in 5 minutes, then with alumina slurry by glass carbon
Glass carbon of the polishing electrode to mirror like surface, successively through ultrasonic cleaning glass-carbon electrode 1min in water, ethyl alcohol, water, after cleaning
H of the electrode in 0.1M2SO4Further electrochemical activation in solution, then with ultrapure water and use N2It is dry；
(4) 1 μ L, 5.0 μM of DNA aptamer drop are handled into gained glassy carbon electrode surface in step (3), 0 DEG C is incubated overnight, and then uses
Deionized water is washed to remove unbonded DNA aptamer；High-specific area nano gold material solution obtained by 5 μ L steps (2) is added dropwise,
High-specific area nano gold material is fixed on glass-carbon electrode by Au-S key and forms aptamer sensor；0- is stored in front of use
In 8 DEG C of refrigerator；
(5) PBS solution for the HepG-2 cell for being the 6.0,0.15M containing concentration by aptamer sensor and pH incubates 3 hours at 20 DEG C
Sandwich shape structure is formed to capture HepG-2 cell (target cell), is then washed 1 time with PBS, electrochemistry aptamer sensor is made；
In Electrochemical Detection, it is placed in the H of quinhydrones and 0.1-10.0mM that 0.1-10mL contains 0.1-10.0mM2O2PBS solution in.
Embodiment two
1, a kind of high-ratio surface nanogold makees the preparation method of the electrochemical sensor of signal amplification carrier, includes the following steps,
(1) it weighs CTAC to be added in 25mL deionized water, adjusting CTAC concentration is 0.6wt%, adds 2.5mL, 30mM
HAuCl4Reaction solution is maintained in 35 DEG C of water-baths by aqueous solution；Then the KBr solution of 50 μ L, 5.0mM is added, stands 20min,
The KI solution of 55 μ L, 5.0mM is then added, then stands 22min, after the L-AA solution for continuously adding 450 μ L, 100mM,
45min is stood to grow Au nanometers of seeds；
(2) by the HAuCl of CTAC and 2.5mL, 30mM4It is added in 25mL deionized water and prepares growth solution, it is dense to adjust CTAC
Degree is 0.6wt%；Be added the l-Glutathione of 5.2 μ L, 50mM into growth solution, be inverted test tube oscillation and be uniformly mixed, then plus
Enter Au nanometers of seed solutions obtained by 55 μ L steps (1), oscillation mixes；Solution is placed in 40 DEG C of water-baths after 45min and is centrifuged richness
Collection washs 2 times, and obtained high-specific area nano gold material is dispersed in 4mL ultrapure water；
(3) glass-carbon electrode is immersed into etching liquid (H2SO4: 30%H2O2=3:1) in 13 minutes, then with alumina slurry by glass
Carbon electrode is polished to mirror like surface, successively through ultrasonic cleaning glass-carbon electrode 1-3min in water, ethyl alcohol, water, after cleaning
H of the glass-carbon electrode in 1.2M2SO4Further electrochemical activation in solution, then with ultrapure water and use N2It is dry；
(4) 15 μ L, 10.0 μM of DNA aptamer drop are handled into gained glassy carbon electrode surface in step (3), 10 DEG C are incubated overnight, so
It is washed with deionized afterwards to remove unbonded DNA aptamer；High-specific area nano gold material obtained by 7 μ L steps (2) is added dropwise
Solution, high-specific area nano gold material are fixed on glass-carbon electrode by Au-S key and form aptamer sensor；Using preceding storage
There are in 0-8 DEG C of refrigerator；
(5) PBS solution for the HepG-2 cell for being the 7.0,0.15M containing concentration by aptamer sensor and pH incubates 5 hours at 38 DEG C
Sandwich shape structure is formed to capture HepG-2 cell, is then washed 2 times with PBS, electrochemistry aptamer sensor is made；In electrochemistry
In detection, it is placed in the H of quinhydrones and 0.1-10.0mM that 0.1-10mL contains 0.1-10.0mM2O2PBS solution in.
Embodiment three
1, a kind of high-ratio surface nanogold makees the preparation method of the electrochemical sensor of signal amplification carrier, includes the following steps,
(1) it weighs CTAC to be added in 50mL deionized water, adjusting CTAC concentration is 0.1wt%, adds 5.0mL, 1mM
HAuCl4Reaction solution is maintained in 50 DEG C of water-baths by aqueous solution；Then the KBr solution of 100 μ L, 0.1mM is added, stands 30min,
The KI solution of 50 μ L, 0.1mM is then added, then stands 30min, it is quiet after the L-AA solution for continuously adding 800 μ L, 5mM
60min is set to grow Au nanometers of seeds；
(2) by the HAuCl of CTAC and 5.0mL, 1mM4It is added in 50mL deionized water and prepares growth solution, adjust CTAC concentration
For 0.1wt%；Be added the l-Glutathione of 10.0 μ L, 0.1mM into growth solution, be inverted test tube oscillation and be uniformly mixed, then plus
Enter Au nanometers of seed solutions obtained by 100.0 μ L steps (1), oscillation mixes；Solution is placed in 50 DEG C of water-baths after -60min and is centrifuged
Enrichment is washed 3 times, and obtained high-specific area nano gold material is dispersed in 5mL ultrapure water；
(3) glass-carbon electrode is immersed into etching liquid (H2SO4: 30%H2O2=3:1) in 20 minutes, then with alumina slurry by glass
Carbon electrode is polished to mirror like surface, the glass successively through ultrasonic cleaning glass-carbon electrode 3min in water, ethyl alcohol, water, after cleaning
H of the carbon electrode in 2.0M2SO4Further electrochemical activation in solution, then with ultrapure water and use N2It is dry；
(4) 25 μ L, 0.1 μM of DNA aptamer drop are handled into gained glassy carbon electrode surface in step (3), 20 DEG C are incubated overnight, then
It is washed with deionized to remove unbonded DNA aptamer；It is molten that high-specific area nano gold material obtained by 10 μ L steps (2) is added dropwise
Liquid, high-specific area nano gold material are fixed on glass-carbon electrode by Au-S key and form aptamer sensor；Using preceding storage
In 0-8 DEG C of refrigerator；
(5) PBS solution for the HepG-2 cell for being the 8.0,0.25M containing concentration by aptamer sensor and pH incubates 1 hour at 50 DEG C
Sandwich shape structure is formed to capture HepG-2 cell, is then washed 3 times with PBS, electrochemistry aptamer sensor is made；In electrochemistry
In detection, it is placed in the H of quinhydrones and 0.1-10.0mM that 0.1-10mL contains 0.1-10.0mM2O2PBS solution in.
Above-described embodiment is related to a kind of octahedra high-specific area nano gold material and the second level superelevation in this, as seed
The preparation of specific surface area nano gold material and the electrochemical sensing for amplifying DNA vector as signal based on this nanogold material
Device preparation, for assessing chlopyrifos and carbofuran to the joint toxicity of cell.Appropriate CTAC is added in ultrapure water, then plus
Enter HAuCl4 aqueous solution, bottle is kept in a water bath.Then 30~60min of a small amount of KBr and KI solution left standstill is sequentially added,
By this bromide and iodide, part replaces chloride to form Au precursor to adjust nanogold pattern and due to the two phase
Reduction potential lower for chloride, can be with relatively quick growth rate into one after reducing agent L-AA is added
One-step growth is octahedra Au nanometers of required seeds.The octahedra nanogold average grain diameter that the invention is prepared is 204.5 nm,
Size is uniform and shape is regular orderly.Likewise, there is the l-Glutathione of chiral conformation, structure by adding in step (2)
The direction of growth special in secondary structure is built out, finally by solution centrifugal enrichment and is dispersed in appropriate ultrapure water.It is this eight
The pattern grown on the basis of the body nanogold of face has thorn-like edge extremely abundant, greatly strengthens biology as nano-carrier
The performance of catalyst.Under certain condition with DNA aptamer reaction bonded, and by product and peroxidase, hemin
It is incubated for, DNA nano-probe of the preparation for signal amplification.Then Thiolation SPECIFIC APTAMER is passed through into the fixed conduct of Au-S key
Modified electrode captures target cell.With the increase of chlopyrifos concentration, differential pulse voltammetry linearly is reduced.
Application Example one
A kind of high-ratio surface nanogold makees the application that the electrochemical sensor of signal amplification carrier evaluate in combined toxicity of pesticides, use
In the detection of chlopyrifos, the electrochemical sensor is electrochemistry aptamer sensor made from the preparation method of above-described embodiment,
The following steps are included: 5 μ L chlopyrifos standard solution or sample solution are added drop-wise on the surface of the electrochemical sensor, 30-
40 DEG C incubation 1-2 hours, washed with PBS and use N2It is dry；Electrochemical sensor is immersed to the phosphate-buffered salt of pH 7.4,10mM
It in solution, is measured using differential pulse voltammetry, sets impulse amplitude 50Mv, the difference arteries and veins being recorded between -0.3V and 0.5V
Rush volt-ampere curve.Prepared aptamer sensor is tested by changing chlopyrifos concentration to respond the DPV of chlopyrifos.Pass through inspection
The reproducibility that 100fM chlopyrifos tests aptamer sensor is surveyed, high duplication is demonstrated.
Application Example two
A kind of high-ratio surface nanogold makees the application that the electrochemical sensor of signal amplification carrier evaluate in combined toxicity of pesticides, use
In the joint toxicity evaluation of binary organophosphorus pesticide on a cellular level, the electrochemical sensor is the preparation of above-described embodiment
Electrochemistry aptamer sensor made from method, comprising the following steps: the HepG-2 cell for taking that treated and growing to logarithmic phase, to
It is wherein added with concentration than the chlopyrifos prepared for 1:1 and carbofuran mixed solution, the chlopyrifos of various concentration and carbofuran exist
It is diluted using preceding with acetone, adds cell culture medium and be formulated into required concentration, and the volumetric concentration of acetone is kept to be lower than 0.5%,
And using the cell base containing 0.5% volumetric concentration acetone as the control group of experiment；Chlopyrifos and carbofuran mixed solution is added in adjustment
Concentration, test electrochemical sensor differential pulse voltammetry response.Since electrochemical sensor has specific sound to chlopyrifos
It answers, joined second of pesticide carbofuran on the basis of the detection of Application Example one, the variation of test sensor response is used to
The effect for evaluating binary pesticide is collaboration antagonism or addition.It is organic phosphorus with other that the above method can be used for chlopyrifos
The joint toxicity evaluation of pesticide on a cellular level.
There are two chief components for electrochemica biological sensor of the invention: the modification of high-specific surface area gold nanoparticle
The glass-carbon electrode interface (GCE) and multi-functional hybridized nanometer probe.Thiolation DNA aptamer can be attached to electrode by Au- S key
Interface is to be used for HepG-2 cell capture.The nano-probe of design is known by the specificity that high-affinity aptamer integrates target cell
Not, and based on high-specific area nano gold electrochemical signals have been expanded.Compared with other pesticide electrochemistry aptamer sensors, it is based on
High-specific area nano gold material has hypersensitivity, high stability as the electrochemical sensor of signal amplification DNA vector
And the advantages of ease for use, while being also used for evaluating the joint toxicity of chlopyrifos and carbofuran in real cell.Wherein, cell is
By poisoning slave girl and carbofuran culture with poison, then it is added drop-wise on the glass-carbon electrode for being modified with functional gold nanoparticles, carries out electrification
Learn measurement.Aptamer sensor is stored two weeks at 4 DEG C can still provide for testing, it is known that it is with high stability.
Claims (6)
1. a kind of high-ratio surface nanogold make signal amplification carrier electrochemical sensor preparation, which is characterized in that including with
Lower step,
(1) it weighs hexadecyltrimethylammonium chloride to be added in 1-50mL deionized water, adjusts hexadecyltrimethylammonium chloride
Concentration is 0.1-1.0wt%, adds the HAuCl of 0.1-5.0mL, 1-30mM4Reaction solution is maintained at 20-50 DEG C of water by aqueous solution
In bath；Then the KBr solution of 5-100 μ L, 0.1-5.0mM is added, stands 10-30min, 5-100 μ L, 0.1- is then added
The KI solution of 5.0mM, then 10-30min is stood, after the L-AA solution for continuously adding 100-800 μ L, 5-100mM, stand
30-60min is to grow Au nanometers of seeds；
(2) by the HAuCl of hexadecyltrimethylammonium chloride and 0.1-5.0mL, 1-30mM4It is added in 1-50mL deionized water
Growth solution is prepared, adjusting hexadecyltrimethylammonium chloride concentration is 0.1-1.0wt%；0.1- is added into growth solution
The l-Glutathione of 10.0 μ L, 0.1-50mM are inverted test tube oscillation and are uniformly mixed, 1.0-100.0 μ L step (1) institute is then added
Au nanometers of seed solutions are obtained, oscillation mixes；Solution is placed in 20-50 DEG C of water-bath centrifugal enrichment after 30-60min, washs 1-3
Secondary, obtained high-specific area nano gold material is dispersed in 3-5mL ultrapure water；
(3) glass-carbon electrode is immersed in etching liquid 5-20 minutes, glass-carbon electrode is then polished to mirror-like with alumina slurry
Surface, successively through ultrasonic cleaning glass-carbon electrode 1-3min in water, ethyl alcohol, water, the glass-carbon electrode after cleaning is in 0.1-2.0M
H2SO4Further electrochemical activation in solution, then with ultrapure water and use N2It is dry；
(4) 1-25 μ L, 0.1-10.0 μM of DNA aptamer drop are handled into gained glassy carbon electrode surface, 0-20 DEG C of incubation in step (3)
Overnight, it is then washed with deionized to remove unbonded DNA aptamer；High-specific surface area obtained by 5-10 μ L step (2) is added dropwise
Nanogold material solution, high-specific area nano gold material are fixed on glass-carbon electrode by Au-S key and form aptamer sensor；
(5) by the PBS solution for the HepG-2 cell that aptamer sensor and pH are 6.0-8.0, the 0.05-0.25M containing concentration in 20-50
It DEG C incubates and to form sandwich shape structure to capture HepG-2 cell in 1-5 hour, then washed 1-3 times with PBS, obtained electrochemistry aptamer
Sensor.
2. high-ratio surface nanogold as described in claim 1 makees the preparation of the electrochemical sensor of signal amplification carrier, special
Sign is that the etching liquid in the step (3) uses H2SO4: 30%H2O2=3:1 etching liquid.
3. high-ratio surface nanogold as described in claim 1 makees the preparation of the electrochemical sensor of signal amplification carrier, special
Sign is that gained aptamer sensor is stored in 0-8 DEG C of refrigerator before use in the step (4).
4. the electrochemistry based on high-specific area nano gold material as signal amplification DNA vector passes as described in claim 1
The preparation method of sensor, which is characterized in that gained electrochemistry aptamer sensor is placed in Electrochemical Detection in the step (5)
0.1-10mL contains the quinhydrones of 0.1-10.0mM and the H of 0.1-10.0mM2O2PBS solution in.
5. a kind of high-ratio surface nanogold makees the application that the electrochemical sensor of signal amplification carrier is evaluated in combined toxicity of pesticides,
It is characterized in that, being used for the detection of chlopyrifos, the electrochemical sensor is using the preparation side as described in claim 1-3 is any
Electrochemistry aptamer sensor made from method, comprising the following steps: be added drop-wise to 5 μ L chlopyrifos standard solution or sample solution described
On the surface of electrochemical sensor, 30-40 DEG C incubation 1-2 hours, washed with PBS and use N2It is dry；Electrochemical sensor is soaked
It in the phosphate buffered saline solution for entering pH 7.4,10mM, is measured using differential pulse voltammetry, sets impulse amplitude 50Mv, note
Record the differential pulse voltammetry curve between -0.3V and 0.5V.
6. a kind of high-ratio surface nanogold makees the application that the electrochemical sensor of signal amplification carrier is evaluated in combined toxicity of pesticides,
It is characterized in that, the joint toxicity evaluation for binary organophosphorus pesticide on a cellular level, the electrochemical sensor are to adopt
The electrochemistry aptamer sensor made from the preparation method as described in claim 1-3 is any, comprising the following steps: take that treated
The HepG-2 cell for growing to logarithmic phase is added thereto with concentration than the chlopyrifos prepared for 1:1 and carbofuran mixed solution,
The chlopyrifos of various concentration and carbofuran are diluted using preceding with acetone, are added cell culture medium and are formulated into required concentration, and
The volumetric concentration of acetone is kept to be lower than 0.5%, and using the cell base containing 0.5% volumetric concentration acetone as the control group of experiment；It adjusts
The whole concentration that chlopyrifos and carbofuran mixed solution is added tests the differential pulse voltammetry response of electrochemical sensor.
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