CN106645129A - Method for detecting chlorpyrifos through functionalized gold nanoparticle based novel colorimetric sensor - Google Patents
Method for detecting chlorpyrifos through functionalized gold nanoparticle based novel colorimetric sensor Download PDFInfo
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- CN106645129A CN106645129A CN201710002777.5A CN201710002777A CN106645129A CN 106645129 A CN106645129 A CN 106645129A CN 201710002777 A CN201710002777 A CN 201710002777A CN 106645129 A CN106645129 A CN 106645129A
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- chlopyrifos
- golden nanometer
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- nanometer particle
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
- G01N2021/3155—Measuring in two spectral ranges, e.g. UV and visible
Abstract
The invention relates to a method for detecting chlorpyrifos through a functionalized gold nanoparticle based novel colorimetric sensor, and belongs to the technical field of analytical chemistry. The method is characterized in that the residual quantity of chlorpyrifos is detected by utilizing the advantages of specific recognition of an aptamer and electrostatic incorporation of a gold nanoparticle; and the method comprises the following detection steps of synthesizing the mercaptoethylamine modified gold nanoparticle; observing the reactions of a system of the gold nanoparticle, the aptamer and the target chlorpyrifos by measuring the ultraviolet visible absorption spectrum; and utilizing the specific binding of the target and the aptamer to detect the chlorpyrifos, and detecting an actual sample. The chlorpyrifos can be simply, rapidly and sensitively detected according to the method, the sensitivity is very high, and the method provides convenience for researches, production, monitoring and the like in the later period.
Description
Technical field
The method that new colorimetric sensor based on functionalization golden nanometer particle detects chlopyrifos, belongs to technique of analytical chemistry
Field.
Background technology
Chlopyrifos is a kind of efficient spectrum insecticide of nineteen sixty-five Dow Chemical Developed, and trade name is
Le Siben, it belongs to heterocyclic organophosphorus pesticide, has become a kind of most large-tonnage organophosphorus pesticide take 40 years.Now by
It is widely used in preventing and treating water paddy and wheat class, vegetables, the insect of flowers and other crops.Chlopyrifos be it is a kind of efficiently, poisoning, broad spectrum activity have
Machine phosphorus insecticide, its toxicity is not very strong, but the skin to human body has strong impulse to act on, enter it is pleasing to the eye in eyes are also had
Certain damage.A small amount of chlopyrifos in higher mammal body can fast degradation be innocuous substance.Poison with poison from from action characteristic
Tick is belonging to nerve toxicant, and its disinfection mechanism is the activity by suppressing biological internal nerve synapse AchE, so as to lead
Cause substantial amounts of AchE to deposit, interrupt nerve conduction, neural paralysis may finally be caused, death is can also result in when serious.
At present, mainly have with regard to the detection method of chlopyrifos both at home and abroad：Gas chromatography, ELISA, and molecule
Engram technology.
Gas chromatography can meet analysis and require, but instrument is typically more costly and complicated, the instrument to operating personnel
The requirement of device operative skill is higher, and at the same time time-consuming, high cost for preparation of samples；Although the more quick letter of ELISA
It is single, but poor anti jamming capability, error of the first kind is there may be to the compound that structure is similar to, and the preparation difficulty of antibody is big,
High cost；Although molecular imprinting sensitivity is high, reaction speed is fast, and selective good, equipment is simple, and it is to reaction condition
Require it is harsh, so this technology is still within laboratory stage, be not widely used in it is actually detected in.Therefore extremely have
Necessity further studies sensitive easy, easily operated, selective height and suitable for the method for actual sample quick detection.
The content of the invention
The purpose of the present invention is the new colorimetric sensor detection chlopyrifos based on functionalization golden nanometer particle, using nucleic acid
Fit specific recognition and its advantage with golden nanometer particle electrostatic adsorption, so as to provide a kind of quick detection chlopyrifos
Method, reach it is quick, simple, it is sensitive detection chlopyrifos residual quantity.
Technical problem：Under new colorimetric sensor based on functionalization golden nanometer particle detects that the method for chlopyrifos is mainly
State principle：
The golden nanometer particle (AuNPs) of mercaptoethylmaine modification is positively charged, and aptamers have electronegative phosphate backbones.Point
Scattered solution of gold nanoparticles is presented claret, when a certain amount of aptamers are added in the solution of gold nanoparticles of positively charged,
Due to electrostatic adsorption, golden nanometer particle is caused to be reunited, solution colour is changed into bluish violet from claret.When chlopyrifos with
Chlopyrifos aptamers combine after will form adaptation composite, the configuration for causing aptamers changes, destruction aptamers with
Electrostatic adsorption between golden nanometer particle, golden nanometer particle is not reunited, and color does not change.Accordingly, can be to poisoning with poison
Tick carries out quantitative determination.
Technical scheme：
Comprise the following steps：The synthesis of the golden nanometer particle of mercaptoethylmaine modification；By determining ultraviolet-visible absorption spectroscopy
The reaction of observation golden nanometer particle, aptamer and object chlopyrifos system；Using object and the specificity of aptamers
With reference to detection chlopyrifos and carry out the detection of actual sample.
(1) synthesis of the golden nanometer particle of mercaptoethylmaine modification
Under conditions of lucifuge, 400 μ L mercaptoethylamine hydrochlorides (0.213M) and 40mL gold chlorides (1.40mM) are added
To in 100mL round-bottomed flasks, 20min is stirred on magnetic stirring apparatus, note lucifuge, new configuration is then rapidly added while stirring
NaBH4Solution 10mL (10mM), continues to stir 30min, finally prepared claret solution, and resulting solution is micro- with 0.45 μm
Hole membrane filtration, filtrate is fitted in brown reagent bottle, preserves in 4 DEG C of refrigerator, standby.
(2) aptamers golden nanometer particle colorimetric detection chlopyrifos method is set up
20 μ L chlopyrifos aptamers (2nM) are added to be then respectively adding 1 μ g/mL different volumes in 1.5mL centrifuge tubes
Chlopyrifos standard liquid, adds distilled water diluting to 500 μ L systems so that the final concentration of chlopyrifos is respectively 1,5,10,20,
40,60ng/mL.After whirlpool mixing 8min, 60 μ L golden nanometer particles (final concentration of 0.077nM) are added, concussion is uniform, reaction
10min, the color of solution obtained by observation, and the ultraviolet-visible absorption spectroscopy of solution is determined with ultraviolet specrophotometer, own
Experiment is carried out at room temperature.
(3) using the specific binding detection chlopyrifos of object and aptamers
To in the mixed system containing 60 μ L AuNPs and 2nM aptamers, the chlopyrifos of variable concentrations is separately added into
Standard liquid, reacts after 8min under room temperature condition, determines the ultraviolet-visible absorption spectroscopy of the system, suctions of the AuNPs at 535nm
Receipts value (A535) will change with the different of chlopyrifos concentration.Respectively with the concentration and A of chlopyrifos535Build for transverse and longitudinal coordinate
Day-mark directrix curve.
The final concentration of chlopyrifos used is followed successively by：1、5、30、50、70、90、100、110ng/mL.In the present invention, poison with poison
The detection of tick is limited to 0.83ng/mL, and the range of linearity is 1-110ng/mL.
(4) measure of selective experiment：
The centrifuge tube of 7 1.5mL is taken, numbering is a～g, is sequentially added into, a～g pipe (60 μ L AuNPs, 2nM nucleic acid
Aptamers and 70ng/mL test substances, test substance organophosphorus pesticide acephatemet, carbamate Methomyl, plan deinsectization successively
Chrysanthemum ester decis and four kinds of analogue Acetamiprids, Atrazine, imidacloprid, 2,4-D butyl esters), determine system 400～
Ultraviolet-visible absorption spectroscopy in the range of 800nm.
(5) detection of actual sample：Taking apple carries out the detection of actual sample
Sample 10g (being accurate to 0.01g) is weighed in 20mL centrifuge tubes, plus 6g anhydrous sodium sulfates and 30mL ethyl acetate,
Homogeneous 2min, in 5000r/min 5min is centrifuged, and in 250mL volumetric flasks, residue adds 30mL ethyl acetate again to supernatant liquid filtering
Extract once, filter, merging filtrate is standby in 250mL volumetric flasks.Method as claimed in claim 4, adds variable concentrations
Chlopyrifos standard liquid be measured.
Beneficial effects of the present invention：
The present invention establishes the method that the new colorimetric sensor based on functionalization golden nanometer particle detects chlopyrifos, the party
Method is simple, quick, sensitive, can quick detection chlopyrifos, it is convenient to provide for supervision from now on.
Description of the drawings
The ultraviolet-visible absorption spectroscopy of Fig. 1 difference systems：a AuNPs；B AuNPs and chlopyrifos aptamers；c AuNPs、
Chlopyrifos, chlopyrifos aptamers；D AuNPs and chlopyrifos
Fig. 2 variable concentrations chlopyrifos (1,5,30,50,70,90,100,110ng/mL) in the presence of golden nanometer particle purple
Outer visible absorption spectra, illustration is corresponding calibration curve
A after different detectable substances is added in Fig. 3 systems535Change：A～g manages (60 μ L AuNPs, 2nM aptamers
With 70ng/mL test substances, test substance organophosphorus pesticide acephatemet, carbamate Methomyl, pyrethroid bromine successively
Cyano chrysanthemate and four kinds of analogue Acetamiprids, Atrazine, imidacloprid, 2,4-D butyl esters)
Fig. 4 golden nanometer particles and the chlopyrifos titer 1 containing variable concentrations, 5,10,30,50,70,100,150,200,
The ultraviolet-visible absorption spectroscopy and working curve diagram of the apple extract system of 250ng/mg.
Specific embodiment
The synthesis of the golden nanometer particle of mercaptoethylmaine modification
Material/agent：Four hydration gold chloride (AuCl3HCl4H2O) sodium borohydride (NaBH4) mercaptoethylamine hydrochloride
(CS) it is purchased from the holding chemical reagent Co., Ltd of Shanghai traditional Chinese medicines
Method：All glasswares needed for experiment soak 24h with chloroazotic acid, with distilled water flushing, and soak 24h, dry
It is standby.The golden nanometer particle synthesis step of mercaptoethylmaine modification is specific as follows：Under conditions of lucifuge, by 400 μ L mercaptoethylmaines
Hydrochloride (0.213M) and 40mL gold chlorides (1.40mM) are added in 100mL round-bottomed flasks, are stirred on magnetic stirring apparatus
20min, notes lucifuge, and NaBH4 solution 10mL (10mM) of new configuration is then rapidly added while stirring, continues to stir 30min,
Final prepared claret solution, by resulting solution with 0.45 μm of filtering with microporous membrane, filtrate is fitted in brown reagent bottle, 4
DEG C refrigerator in preserve, it is standby.
As a result：Obtain the solution of gold nanoparticles of the mercaptoethylmaine modification of claret
Because the volume of solution of gold nanoparticles can produce certain impact to experimental system, it is therefore desirable to study gold nano
The action effect of the volume of particle.
Material/agent：Solution of gold nanoparticles：Oneself synthesizes according to document
Method：The centrifuge tube of 6 1.5mL is taken, a～f is numbered, the chlopyrifos aptamers of 2nM is added in each pipe, so
It is separately added into again afterwards：40th, 50,60,70,80,90 μ L solution of gold nanoparticles, after reaction completely, determines the ultraviolet of above-mentioned solution
Visible absorption spectra, calculates A535Drop-out value.
As a result：With the increase of solution of gold nanoparticles concentration, A535Drop-out value can first increase and reduce afterwards, when adding gold
When nano-particle solution volume is 60 μ L, the value reaches maximum, and experimental phenomena is most obvious, therefore, solution of gold nanoparticles is most
Good volume is 60 μ L.
In addition, the concentration of aptamer also can produce certain impact to experimental system, it is therefore desirable to which research is suitable
The action effect of ligand concentration.Material/agent：Chlopyrifos aptamer：Synthesized by raw work biology (Shanghai) Reagent Company
Method：The centrifuge tube of 6 1.5mL is taken, a～f is numbered, 60 μ L solution of gold nanoparticles is added in each pipe,
Then it is separately added into again：0.2nd, 0.6,1.0,2.0,3.0,4.0nM chlopyrifos aptamers, the polishing that adds water is to 500 μ L, and reaction is certain
After time, the ultraviolet-visible absorption spectroscopy of above-mentioned solution is determined, meanwhile, in another group of centrifuge tube, add the μ g/mL of 50 μ L 1 malicious
Dead tick titer (final concentration 100ng/mL), is added thereto to the chlopyrifos aptamers with upper one group of same volume, is vortexed certain
Time makes it fully combine, and adds 60 μ L solution of gold nanoparticles and the polishing that adds water is to 500 μ L, after reaction certain hour, surveys
Ultraviolet-visible absorption spectroscopy is determined, by the A for obtaining twice535Do difference.
As a result：Chlopyrifos is adapted to bulk concentration in below 2nM, and difference gradually increases, and reaches peak value in 2nM, when dense
When degree is more than 2nM, difference is still than larger, but due to aptamers excessive concentration can affect the sensitivity tested, therefore, adaptation
The final concentration of body elects 2nM as.
In addition, golden nanometer particle also can produce action effect with the chlopyrifos aptamers reaction time to experiment, therefore
Need to be optimized its reaction time.
Material/agent：Solution of gold nanoparticles：Oneself synthesizes according to document；Chlopyrifos aptamer：It is biological by raw work
(Shanghai) Reagent Company synthetic method：Take and be separately added in one group of 1.5mL centrifuge tube 60 μ L solution of gold nanoparticles and 20 μ L
50nM chlopyrifos aptamers, the benefit that adds water to 500 μ L systems, after different time (1-8min) is reacted respectively, determine its it is ultraviolet can
See absorption spectrum.
As a result：Analysis understands that golden nanometer particle is 7min with chlopyrifos aptamers optimum reacting time.
In addition, chlopyrifos and chlopyrifos aptamers reaction time also can be to experiment generation action effects, it is therefore desirable to
Its reaction time is optimized.
Material/agent：Solution of gold nanoparticles：Oneself synthesizes according to document；Chlopyrifos aptamer：It is biological by raw work
(Shanghai) Reagent Company synthesizes；Chlopyrifos：Buy in Sigma-Aldrich Reagent Company
Method：One group of 1.5mL centrifuge tube is taken, 20 μ L chlopyrifos aptamers are separately added into, 50 μ L chlopyrifos standard liquids, often
The individual centrifuge tube vortex mixed different time (1-10min), it is separately added into the μ L of solution of gold nanoparticles 60, and with water polishing extremely
500 μ L systems, after reaction 7min its ultraviolet-visible absorption spectroscopy is determined.
As a result：Analysis understands that chlopyrifos is 8min with the optimum reacting time of chlopyrifos aptamers.
The method is for chlopyrifos detection：
Material/agent：Solution of gold nanoparticles：Oneself synthesizes according to document；Chlopyrifos aptamer：It is biological by raw work
(Shanghai) Reagent Company synthesizes；Chlopyrifos is purchased from Sigma-Aldrich Reagent Company；Apple is purchased from local food market
Method：
(1) using aptamers golden nanometer particle colorimetric determination chlopyrifos：To containing 60 μ L solution of gold nanoparticles, 2nM
In the mixed system of aptamer, the chlopyrifos standard liquid of variable concentrations is separately added into, is reacted under room temperature condition after 8min,
The ultraviolet-visible absorption spectroscopy of the system is determined, absorbance of the golden nanometer particle at 535nm is by with chlopyrifos concentration
It is different and change.
(2) the chlopyrifos standard concentration used by is followed successively by：1st, 5,30,50,70,90,100,110ng/mL, by determining
Absorbance A of the golden nanometer particle at 535nm535, according to A535Calibration curve is set up with the concentration of chlopyrifos.
(3) detection of actual sample：Taking apple carries out the detection of actual sample
Sample 10g (being accurate to 0.01g) is weighed in 20mL centrifuge tubes, plus 6g anhydrous sodium sulfates and 30mL ethyl acetate,
Homogeneous 2min, in 5000r/min 5min is centrifuged, and in 250mL volumetric flasks, residue adds 30mL ethyl acetate again to supernatant liquid filtering
Extract once, filter, merging filtrate is standby in 250mL volumetric flasks.Method as claimed in claim 4, adds variable concentrations
Chlopyrifos standard liquid be measured.
(4) checking of experimental system：
Selective experiment：
The centrifuge tube of 8 1.5mL is taken, numbering is a～h, is sequentially added into, a～h pipes (60 μ L solution of gold nanoparticles,
2nM aptamers, 70ng/mL test substances, test substance is followed successively by organophosphorus pesticide acephatemet, carbamate and goes out many
Prestige, pyrethroid decis and four kinds of analogue Acetamiprids, Atrazine, imidacloprid, 2,4-D butyl esters and poison with poison
Tick), determine the absorbance A at 535nm of above-mentioned solution535。
As a result：Respectively with the concentration and A of chlopyrifos535Calibration curve is set up for transverse and longitudinal coordinate, detection is calculated and is limited to
0.83ng/mL, the range of linearity is 1-110ng/mL.
The mark-on reclaims result of the test of this experimental technique detection chlopyrifos see the table below
SEQUENCE LISTING
<110>Jilin University
<120>New colorimetric sensor based on functionalization golden nanometer particle detects chlopyrifos
<160> 1
<210> 1
<211> 90
<212> DNA
<213>Artificial sequence
<400> 1
cctgccacgt ccgcaagctt agggttacgc ctgcagcgat tcttgatcgc gctgctggta
atccttcttt aagcttggca cccgcatcgt 90
Claims (5)
1. the new colorimetric sensor detection chlopyrifos based on functionalization golden nanometer particle, is characterized in that using the spy of aptamer
Opposite sex identification and its advantage with golden nanometer particle electrostatic adsorption, in building a kind of new colorimetric sensor to detect fruits and vegetables
The residual of chlopyrifos, comprises the following steps：The synthesis of the positive electricity golden nanometer particle (CS-AuNPs) of mercaptoethylmaine modification；By surveying
Determine the reaction of ultraviolet-visible absorption spectroscopy observation golden nanometer particle, aptamer and object chlopyrifos system；Using target
Thing detects chlopyrifos and carries out the detection of actual sample with the specific binding of aptamers.
2. the method for claim 1, wherein the positive electricity golden nanometer particle (CS-AuNPs) of mercaptoethylmaine modification
Synthesis step is as follows：
Under conditions of lucifuge, 400 μ L mercaptoethylamine hydrochlorides (0.213M) and 40mL gold chlorides (1.40mM) are added to
In 100mL round-bottomed flasks, 20min is stirred on magnetic stirring apparatus, note lucifuge, new configuration is then rapidly added while stirring
NaBH4Solution 10mL (10mM), continues to stir 30min, finally prepared claret solution, by resulting solution with 0.45 μm of micropore
Membrane filtration, filtrate is fitted in brown reagent bottle, preserves in 4 DEG C of refrigerator, standby.
3. the method for claim 1, wherein described observe golden nanometer particle, core by determining ultraviolet-visible absorption spectroscopy
The step of reaction of sour aptamers and object chlopyrifos system, is as follows：
20 μ L chlopyrifos aptamers (2nM) are added to be then respectively adding poisoning with poison for 1 μ g/mL different volumes in 1.5mL centrifuge tubes
Tick standard liquid, adds distilled water diluting to 500 μ L systems so that the final concentration of chlopyrifos is respectively 1,5,10,20,40,
60ng/mL；After whirlpool mixing 8min, 60 μ L golden nanometer particles (final concentration of 0.077nM) are added, concussion is uniform, reaction
10min, the color of solution obtained by observation, and the ultraviolet-visible absorption spectroscopy of solution is determined with ultraviolet specrophotometer, own
Experiment is carried out at room temperature.
4. the method for claim 1, wherein the specific binding detection chlopyrifos of the utilization object and aptamers
The step of it is as follows：
To in the mixed system containing 60 μ L AuNPs and 2nM aptamers, the chlopyrifos standard of variable concentrations is separately added into
Solution, reacts after 8min under room temperature condition, determines the ultraviolet-visible absorption spectroscopy of the system, suctions of the CS-AuNPs at 535nm
Receipts value (A535) will change with the different of chlopyrifos concentration.
5. the method for claim 1, wherein the detection of the actual sample is to take apple extract to carry out：Weigh sample
10g (being accurate to 0.01g) is in 20mL centrifuge tubes, plus 6g anhydrous sodium sulfates and 30mL ethyl acetate, homogeneous 2min, in 5000r/
Min is centrifuged 5min, and in 250mL volumetric flasks, residue adds 30mL ethyl acetate to extract again once to supernatant liquid filtering, filters, and closes
Standby and filtrate is in 250mL volumetric flasks, method as claimed in claim 4 adds the chlopyrifos standard liquid of variable concentrations
It is measured, respectively with the concentration and A of chlopyrifos535Calibration curve is set up for transverse and longitudinal coordinate, the detection for calculating chlopyrifos is limited to
0.83ng/mL, the range of linearity is 1-110ng/mL.
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| CN107478647A (en) * | 2017-08-17 | 2017-12-15 | 安庆师范大学 | A kind of method based on noble metal nano particles rapid field detection decis |
| CN110125437A (en) * | 2019-05-29 | 2019-08-16 | 浙江工业大学 | A kind of preparation method of the size tunable gold nano grain of positive charge modification |
| CN113959968A (en) * | 2021-11-18 | 2022-01-21 | 南京林业大学 | Method for detecting microcystin MC-LR by ratio type colorimetric aptamer sensor based on AuNPs |
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| CN113959968A (en) * | 2021-11-18 | 2022-01-21 | 南京林业大学 | Method for detecting microcystin MC-LR by ratio type colorimetric aptamer sensor based on AuNPs |
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Application publication date: 20170510 |