CN103063842A - Preparation method of immunosensor for detecting Carbofuran pesticide residues - Google Patents
Preparation method of immunosensor for detecting Carbofuran pesticide residues Download PDFInfo
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- CN103063842A CN103063842A CN2013100006639A CN201310000663A CN103063842A CN 103063842 A CN103063842 A CN 103063842A CN 2013100006639 A CN2013100006639 A CN 2013100006639A CN 201310000663 A CN201310000663 A CN 201310000663A CN 103063842 A CN103063842 A CN 103063842A
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Abstract
The invention relates to a preparation method of an immunosensor for detecting Carbofuran pesticide residues. The preparation method is characterized by comprising the following steps: self-assembling 4' 4-glucosinolates biphenyl thiophenol, carrying out electrolytic deposition on nanogold and repeating the first step and the second step twice; and compared with a traditional method, sulfydryl at the two ends of the 4' 4-glucosinolates biphenyl thiophenol and a rigid structure of the 4'4-glucosinolates biphenyl thiophenol are adopted, one end of the sulfydryl is connected with a bare gold electrode through a gold-sulfur bond, the other end of the sulfydryl is connected with the nanogold subjected to electrolytic deposition through the gold-sulfur bond, and a gold-sulfur system with high stability and high order is established through a layer-by-layer self-assembly method. The preparation method has the benefits that due to higher specific surface area, stability and electrical conductivity of the nanogold, the transfer and the biocompatibility of an electrode surface electron can be increased; through layer-by-layer self-assembly, the effective fixed area of the electrode is enlarged, and the effective fixed amount of an electrode surface antibody is improved; and therefore, the prepared current type immunosensor has the advantages of shorter detection time, high sensitivity, good selectivity and stability, good regeneration capacity and satisfactory recovery rate.
Description
Technical field
The invention provides a kind of immunosensor preparation method who detects the agricultural chemicals Determination of carbofuran, belong to the biosensor technology field.
Background technology
In recent years, the poisoning that residues of pesticides cause occurs repeatedly, and the residue problem harmful, highly toxic pesticide in the fruits and vegetables is the focus that people pay close attention to always, also be now China's entry into the WTO after, the export of farm produce meets with one of the major influence factors of " green barrier ".Carbofuran is one of carbamate insecticides principal item, and it is widely used in the current agricultural as a kind of spectrum pesticide.This medicament has interior absorption, can pass through root, stem, leaf or seed Assimilation and conductivity, and is transported to each organ of plant, also can pass through runoff osmosis pollution underground water.Research finds that the metabolin of carbofuran can cause the dna single chain fracture that cell is strong, has obvious cytotoxicity.Although on vegetables, forbidden in the world in recent years this agricultural chemicals, because it has growth stimulation to some crop, so still have many phenomenons of using violating the regulations.Have the fruits and vegetables that highly toxic like this residues of pesticides exceed standard and come into the market, will be to the health of people harm, therefore set up a kind of fast, sensitive, safe and reliable Detecting Pesticide analytical approach is extremely urgent.
Traditional analytical approach for detection of Determination of carbofuran has: gas chromatography (GC), high performance liquid chromatography (HPLC), Chromatography/Mass Spectrometry coupling technique (GC/LC-MS), capillary electrophoresis (CE), fluorescence analysis, enzyme linked immunological (ELISA) etc., although these traditional methods have high selectivity and sensitivity, but the restriction of the factors such as it is subject to, and sample pre-treatments is consuming time, instrument and equipment is expensive, operating personnel's technical requirement high, detection time is long, laboratory operation can only be confined to, the requirement that the on-the-spot speed of fruits and vegetables is surveyed can not be satisfied.Immunosensor is the sensor that is developed into according to the immunoreactive principle that antigen and antibody specific binding cause, and compares with traditional analytical approach, and it has the advantages such as high specificity, analysis speed be fast, simple in structure, with low cost.And in the preparation process of immunosensor, increase electric current response, antigen/antibody immobilization and to keep its good biologically active and spatial arrangement be the committed step of restriction sensor preparation, these will directly affect the main performances such as stability, reappearance, sensitivity and selectivity of immunosensor.
The purpose of invention is to provide a kind of and can overcomes defects, simple to operate, highly sensitive, the preparation method of the current mode immunosensor of the detection agricultural chemicals Determination of carbofuran that selectivity is good.
Its technical scheme is: a kind of immunosensor preparation method who detects the carbofuran residues of pesticides, it is characterized in that: the assembling process of current mode immunosensor is self assembly 4 ' 4-sulfo-biphenyl thiophenol (DMDPSE), electro-deposition nm of gold (DpAu), repeat the first step and second step 2 times, make up stable golden sulfur system, then fixing carbofuran monoclonal antibody, finish with the preparation of BSA sealing current type immunosensor at last, put in the refrigerator 4 ℃ and save backup.
Described a kind of immunosensor preparation method who detects the carbofuran residues of pesticides is characterized in that: the optimization of experiment condition mainly comprises the number of plies of electro-deposition nm of gold, pH and the incubation time of test end liquid; The working curve of prepared current mode immunosensor is: % Δ I=16.676+8.7332 lgC (ng/mL); The immunosensor Performance Detection comprises that specificity, stability, reappearance, reproducibility and immunosensor are to the mensuration of the vegetable sample recovery.
Its preparation principle is: the present invention adopts 4 ' 4-sulfo-biphenyl thiophenol, electro-deposition nm of gold that gold electrode is modified, compare with traditional method, the sulfydryl that adopts 4 ' 4-sulfo-biphenyl thiophenol two ends with and rigid structure, sulfydryl one end connects naked gold electrode by golden sulfide linkage, the sulfydryl other end connects the nm of gold of electro-deposition by golden sulfide linkage, and the method by layer by layer self assembly, made up a kind of golden sulphur (Au-S) system with high stability, high-sequential.Because higher specific surface area, stability and the electric conductivity of nm of gold can increase transfer and the biocompatibility of electrode surface electronics, and then increase the response current of immunosensor; Compare with traditional method that is coated with the nanometer gold size of dripping, the nm of gold of electro-deposition has better stability, uniformity at electrode surface, and deposition process simple, save time, controlled.The nm of gold of electro-deposition for antibody fixedly providing of electrode surface a good bioelectric interface, the biologically active that has kept antibody is to be used for and the effective specific binding of agricultural chemicals.By layer by layer self assembly, enlarged effective fixed-area of electrode, improved effective fixed amount of electrode surface antibody.After will making immunosensor through the gold electrode of above-mentioned steps modified, the purpose that the precision that detects the agricultural chemicals carbofuran is higher, realization is small-sized, convenient, be applicable to Site Detection.Before the current mode immunosensor that adopts the present invention to make can be gathered, go on the market at vegetables and fruits, carry out the Fast Measurement of agricultural chemicals Determination of carbofuran, directly whether superscale detects to the carbofuran residues of pesticides, the vegetables and fruits of avoiding containing remains of pesticide because of food cause poisoning, and the technical support of residue detection is provided for crop production safety and consumption.
For reaching above purpose, take following technical scheme to realize: a kind of immunosensor preparation method who detects the carbofuran residues of pesticides, it is characterized in that: ⑴ disposes a series of carbofuran titer, in order to working curve, sensing range and the detectability of the current mode immunosensor that draws above-mentioned preparation; ⑵ dispose the pesticidal solutions that a series of frequent mixing are used, to detect the selectivity of prepared current mode immunosensor;
Verify the stability of above-mentioned immunosensor by the cyclic voltammetry scan multistage, by dissociated antigen antibody mediated immunity compound and its regenerability of immune detection again;
Draw the recovery of standard addition of immunosensor by detecting actual vegetable sample.
It is self-assembled modified that the present invention adopts two thiol compound 4 ' the 4-sulfo-biphenyl thiophenols of rigidity, the electro-deposition nm of gold is carried out electrode surface, compare with the method for traditional self-assembled nanometer gold size and to have increased its stability, fixed amount, the golden sulfur system that forms has higher stability and high-sequential, increase the current-responsive of sensor and improved the effectively fixing of sensor surface antibody, thereby increased accuracy of detection.
The preparation technology of described current mode immunosensor is as follows: lucifuge is assembled 24h in the ethanolic solution of 2.0 mM, 4 ' the 4-sulfo-biphenyl thiophenol of the gold electrode immersion letting nitrogen in and deoxidizing that ⑴ is good with pre-service, and dries up with nitrogen.In the chlorauric acid solution that contains 0.1M potassium chloride of 5mL 0.3mM, under the voltage of-0.2V, use potentiostatic method electro-deposition 200s.With the PBS damping fluid flushing of PH=7.5, nitrogen dried up after electro-deposition finished.
Repeating step one and step 2 n time;
The above-mentioned electrode for preparing is dipped in the carbofuran monoclonal antibody solution, soaks 12h under 4 ℃ of conditions, wash away redundance after the taking-up, dry stand-by;
With leaving standstill 1h under the room temperature in the BSA solution of the last immersion 2.5% of the above-mentioned electrode for preparing, with nonspecific binding site on the enclosed-electrode, take out to place under the room temperature and dries at last, immunosensor completes, and is kept under 4 ℃ of conditions for subsequent use.
Embodiment
Embodiment: the cleaning of the naked gold electrode of ⑴: before described naked gold electrode is modified, at first use the Al of 0.3 μ m, 50nm
2O
3Slurry is polished to minute surface at chamois leather, with deionized water flush away surface contaminants, moves in the ultrasonic water bath and cleans after the polishing, and each 5min repeats secondary, then uses successively the HNO of 6M
3, absolute ethyl alcohol and deionized water ultrasonic cleaning, nitrogen dries up;
The activation of naked gold electrode: thoroughly after the washing, electrode is at 0.5M H
2SO
4Activate with cyclic voltammetry in the solution, sweep limit-0.3～1.5V, scanning is until till reaching stable cyclic voltammogram repeatedly.
Lucifuge assembling 24h(is denoted as DMDPSE/Au in the ethanolic solution of 2.0 mM, 4 ' the 4-sulfo-biphenyl thiophenol of the gold electrode immersion letting nitrogen in and deoxidizing that pre-service is good), take out electrode, wash 4 ' the 4-sulfo-biphenyl thiophenol that electrode surface does not adsorb with removal with absolute ethyl alcohol, and dry up with nitrogen.
In the chlorauric acid solution that contains 0.1M potassium chloride of 5mL 0.3mM, under the voltage of-0.2V, use potentiostatic method electro-deposition 200s(and be denoted as DpAu/DMDPSE/Au), with the PBS damping fluid flushing of PH=7.5, nitrogen dried up after electro-deposition finished.
Repeating step one and step 2 (are denoted as { DpAu/DMDPSE} 2 times
2/ Au);
The above-mentioned electrode for preparing is dipped in the carbofuran monoclonal antibody solution, soaks 8h under 4 ℃ of conditions, wash away redundance after the taking-up, dry stand-by;
To leave standstill 2h under the room temperature in the BSA solution of the last immersion 1% of the above-mentioned electrode for preparing at last, with nonspecific binding site on the enclosed-electrode, taking-up places under the room temperature dries, and immunosensor completes and (is denoted as BSA/anti-carbofuran/ { DpAu/DMDPSE }
n/ Au), be kept under 4 ℃ of conditions for subsequent use;
Be optimized from the electro-deposition decorated by nano-gold number of plies, pH and the incubation time three aspects: experiment condition to prepared immunosensor, the modification number of plies of nm of gold is 1-3, and the scope of pH is 5.0-9.0, and the scope of incubation time is 10-70min.
Configuration 0.1-1.0 * 10
6The carbofuran standard solution of ng/mL immerses respectively the carbofuran standard solution of variable concentrations with the above-mentioned current mode immunosensor for preparing, and hatches at normal temperatures 30min, detect immunity before and after curent change obtain its working curve;
With the current mode immunosensor respectively 1.0 * 10
3Ng/mL often mixes in the pesticidal solutions of use hatches 40min under the normal temperature, to detect its selectivity; The immunosensor that selects the six roots of sensation to prepare detects its reappearance; The continuous carbofuran solution of same concentrations that detected successively in 7 days is to detect its stability; Sensor after the immunity with glycocoll-HCl damping fluid (pH 2.8) again immunity behind the carbofuran agricultural chemicals 5min of dissociating, is detected its power of regeneration;
Vegetables are thoroughly cleaned up and with washed with de-ionized water 3 times, certain density agricultural chemicals in the sprinkling, place 24h after, do solvent supersonic with PBS (pH 7.5) and process 30min (changing solvent three times), with the recovery of detection actual sample.
The detection method operating procedure that this kind immunosensor detects the agricultural chemicals Determination of carbofuran is simple, detection time is shorter, and is highly sensitive, and selectivity and stability is good, good and the recovery of power of regeneration meets the requirements, and meets China's Fast Determination of Pesticide Residue technical development and internationalization requirement.
Claims (3)
1. immunosensor preparation method who detects the carbofuran residues of pesticides, it is characterized in that: the group shape process of current mode immunosensor is self assembly 4 ' 4-sulfo-biphenyl thiophenol, electro-deposition nm of gold, repeat the first step and second step 2 times, self assembly carbofuran monoclonal antibody, self assembly BSA sealing.
2. a kind of immunosensor preparation method who detects the carbofuran residues of pesticides as claimed in claim 1, it is characterized in that: utilize the good electric conductivity of nm of gold, large specific surface area and biocompatibility to be used for improving electrode performance and adhesion protein A, utilize albumin A to come directional immobilizing antibody with the Fc terminal specific combination of antibody, with the more effective immune detection of finishing the target component carbofuran.
3. a kind of immunosensor preparation method who detects the carbofuran residues of pesticides as claimed in claim 1, the modification of immunosensor and characterization, the optimization of relevant experiment parameter, the working curve of prepared immunosensor is: carbofuran concentration 0.1-10
6During ng/mL, the working curve that detects the carbofuran agricultural chemicals is % Δ I=16.676+8.7332 lgC (ng/mL), detects and is limited to 0.06ng/mL.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103454426A (en) * | 2013-09-10 | 2013-12-18 | 山东理工大学 | Preparation method of nanogold/chitosan-graphene-methylene blue modified immunosensor |
| CN103940868A (en) * | 2014-04-15 | 2014-07-23 | 山东理工大学 | Fast detector of pesticide residues based on enzyme immunosensor |
| CN104630869A (en) * | 2015-01-22 | 2015-05-20 | 江南大学 | DNA sensor for detecting staphylococcus aureus as well as preparation method and application of DNA sensor |
| CN104914238A (en) * | 2015-06-11 | 2015-09-16 | 山东理工大学 | Preparation method of microfluidic immunosensor |
| CN106370708A (en) * | 2016-08-29 | 2017-02-01 | 南京师范大学 | Electrochemistry sensor for measuring organophosphorus pesticide residues and measurement method thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103454426A (en) * | 2013-09-10 | 2013-12-18 | 山东理工大学 | Preparation method of nanogold/chitosan-graphene-methylene blue modified immunosensor |
| CN103454426B (en) * | 2013-09-10 | 2015-07-01 | 山东理工大学 | Preparation method of nanogold/chitosan-graphene-methylene blue modified immunosensor |
| CN103940868A (en) * | 2014-04-15 | 2014-07-23 | 山东理工大学 | Fast detector of pesticide residues based on enzyme immunosensor |
| CN103940868B (en) * | 2014-04-15 | 2016-02-24 | 山东理工大学 | A kind of rapid detector for pesticide residue based on enzyme immunosensor |
| CN104630869A (en) * | 2015-01-22 | 2015-05-20 | 江南大学 | DNA sensor for detecting staphylococcus aureus as well as preparation method and application of DNA sensor |
| CN104630869B (en) * | 2015-01-22 | 2017-07-14 | 江南大学 | A kind of DNA sensor for detecting staphylococcus aureus and its preparation and application |
| CN104914238A (en) * | 2015-06-11 | 2015-09-16 | 山东理工大学 | Preparation method of microfluidic immunosensor |
| CN106370708A (en) * | 2016-08-29 | 2017-02-01 | 南京师范大学 | Electrochemistry sensor for measuring organophosphorus pesticide residues and measurement method thereof |
| CN106370708B (en) * | 2016-08-29 | 2018-09-14 | 南京师范大学 | A kind of electrochemical sensor and its detection method of organophosphorus pesticide residual quantity detection |
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Application publication date: 20130424 |