CN102879575A - Preparation method for immunosensor based on antibody directional fixation and nano-composite material - Google Patents
Preparation method for immunosensor based on antibody directional fixation and nano-composite material Download PDFInfo
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Abstract
The invention relates to a preparation method for an immunosensor based on antibody directional fixation and a nano-composite material. The preparation method is characterized in that glutathione is used as a spatial arm to directionally and covalently fix monoclonal antibody of an anti-carbofuran pesticide on the surface of colloidal gold nanoparticle, wherein the bioactivity of the antibody and a free antigen binding site are kept by the directional fixation of the antibody to be effectively and specifically combined with the pesticide, so that the detection sensitivity of the sensor is enhanced. A novel nanomaterial GS-PEI-Au nano-composite material is prepared for improving the water solubility, the conductivity and the stability of graphene. An electrode is modified by synergistic effects of two nano-composite materials including MWCNTs-CS and GS-PEI-Au together, so that the current response is obviously enhanced, the effective fixed area of the electrode is expanded, and the effective fixation quantity of the antibody on the surface of the electrode is improved. After the electrode modified by the steps is manufactured into the immunosensor, the detection time is shorter, the sensitivity is high, the selective stability is high, the regeneration capacity is high, and the recovery rate meets the requirements.
Description
Technical field
The invention provides a kind of immunosensor preparation method of and nano composite material directed fixing based on antibody, 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 residues of pesticides 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 is 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.In the preparation process of current mode immunosensor, the immobilization of antibody (Ab) or haptens (hapten) and to keep the amplification of its good biologically active, spatial arrangement and signal be the key factor of restriction transducer sensitivity, stability, selectivity and regenerability etc.
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 residues of pesticides that selectivity is good.
Its technical scheme is: a kind of immunosensor preparation method of and nano composite material directed fixing based on antibody, it is characterized in that: realize that antibody is directed fixing, the monoclonal antibody of anti-carbofuran (Ab) is directed covalency with glutathione (glutathione, G-SH) as the space arm and is fixed to nanometer gold size surface.
The immunosensor preparation method of described a kind of and nano composite material directed fixing based on antibody, it is characterized in that: prepare respectively many walls carbon-shitosan (MWCNTs-CS), two kinds of nano composite materials of Graphene-polyethyleneimine-nm of gold (GS-PEI-Au), and the common modified electrode of the synergy of utilizing these two kinds of nano materials, to amplify response current.
The immunosensor preparation method of described a kind of and nano composite material directed fixing based on antibody is characterized in that: the optimization of experiment condition mainly comprises pH, incubation temperature and the incubation time of test end liquid; The working curve of prepared immunosensor is: % Δ I=14.275+11.087 lgC (ng/mL), (R
2=0.9889.), its detection is limited to 0.03ng/mL; The immunosensor Performance Detection comprises that specificity, stability, reappearance, reproducibility and immunosensor are to the mensuration of multiple fruits and vegetables sample recovery rate.
Its preparation principle is: the present invention adopts glutathione (glutathione, G-SH) will resist the directed covalency of monoclonal antibody (Ab) of carbofuran to be fixed to nanometer gold size surface as the space arm, wherein G-SH firmly is fixed to nanometer gold size surface by golden sulfide linkage, G-SH terminal carboxyl group (– COOH) through 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (EDC) and 1, after 6-hexane diamine (DAH) activation, the – COOH of covalent bond antibody C end, thus realized that the Ab orientation is connected to nanometer gold size surface.The orientation of antibody fixedly kept the biologically active of antibody and freely antigen binding site come and the effective specific binding of agricultural chemicals.Adopt many walls carbon-shitosan (MWCNTs-CS), two kinds of common modified electrodes of nano composite material of Graphene-polyethyleneimine-nm of gold (GS-PEI-Au), strengthened significantly current-responsive, enlarge effective fixed-area of electrode, improved effective fixed amount of electrode surface antibody.After will making immunosensor through the electrode of above-mentioned steps modified, the purpose that the precision that detects agricultural chemicals is higher, realization is small-sized, convenient, be applicable to Site Detection.Before the 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 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 based on the directed fixing and nano composite material of antibody, it is characterized in that: ⑴ by covalent approach with the antibody orientation be fixed on nanometer gold size surface, the orientation of antibody fixedly kept the biologically active of antibody and freely antigen binding site come and the effective specific binding of agricultural chemicals; ⑵ based on the synergy of MWCNTs-CS and two kinds of nano composite materials of GS-PEI-Au, with its common modified electrode.
The present invention has prepared a kind of novel nano-material GS-PEI-Au nano composite material, is used for improving water-soluble, electric conductivity and the stability of GS.Adopt the common modified electrode of synergy of MWCNTs-CS and two kinds of composite nano materials of GS-PEI-Au, amplify response signal, the useful area that increases electrode is used for fixing more antibody.In addition, the orientation by antibody has fixedly kept the biologically active of antibody and antigen binding site freely, strengthens the detection sensitivity of sensor.
The preparation technology of described current mode immunosensor is as follows: ⑴ prepares MWCNTs-CS nano composite material, GS-PEI-Au nano composite material and AuNPs-Ab conjugate;
The MWCNTs-CS nano composite material is modified cleaned naked glass-carbon electrode modify oven dry;
Modify the GS-PEI-Au nano composite material, oven dry;
Modify the AuNPs-Ab conjugate, hatch 12h for 4 ℃; ⑹, dry under the room temperature with nonspecific binding site on the enclosed-electrode leaving standstill 30min under the room temperature in the BSA solution of the last immersion 1% of the above-mentioned electrode for preparing, and immunosensor completes, and is kept under 4 ℃ of conditions for subsequent use.
Embodiment
Embodiment: the preparation of ⑴ MWCNTs-CS nano composite material: shitosan (CS) is dissolved in 1.0% the acetum, is made into 0.25% CS solution, the room temperature lower magnetic force stirs 1h, and shitosan is dissolved fully, then with NaOH the pH of mixed liquor is transferred to 5.0.Take by weighing 1mg MWNTs, add 1mL 0.25% chitosan solution, ultrasonic dispersion 2h obtains stable black dispersion liquid (MWCNTs-CS nano composite material) at last again.
The preparation of GS-PEI-Au nano composite material: take by weighing 3mg GS, add the 10mL deionized water, ultrasonic dispersion 10min obtains the GS dispersion liquid.Add 0.2 mL, 1.0 M PEI aqueous solution in the GS dispersion liquid, then ultrasonic dispersion 10min adds 1.6 mL 21mM HAuCl
4, with 70 ℃ of water-bath 2h of this mixed liquor, take out, be cooled to room temperature.Centrifugal 10 min of 15,000 rpm remove supernatant, and are repeatedly centrifugal, and centrifugal gained sediment is dissolved in the 1.5 mL deionized waters, and ultrasonic dispersion 5min obtains stable black dispersion liquid (GS-PEI-Au nano composite material).This dispersion liquid at room temperature can keep one month stability.
The preparation of AuNPs-Ab conjugate: get the AuNPs solution that 5 mL prepare, add the G-SH of 4 mL, 65 μ M, stir 10-15min, mixed liquor is in the centrifugal 30min of 4500 rpm, and centrifugal product is suspended among the 1 mL PBS, obtains the AuNPs that G-SH modifies.In this product, add 300 μ L, 58 mM EDC and 3 mL f MES buffer solution (pH 5.0), 4 ℃ of lower 1.5 h that place in a cantilever arrangement, then in 15, centrifugal 1 h isolates the AuNPs(EDC/AuNPs that EDC has activated under 000 rpm), this reactant is suspended among the 1 mL PBS.Add 50 μ L, 500 μ M DAH in this reactant, mix 30min under the room temperature, the reaction mixture of gained is again centrifugal, obtains the AuNPs (DAH/EDC/AuNPs) that DAH has activated, and is suspended among the 1 mL PBS.Get 500 μ L, 10 μ g/mL Ab and add 3 mL MES buffer solution (pH 5.0) and 300 μ L, 58 mM EDC, 4 ℃ of lower 1.5 h that place in a cantilever arrangement.Ab after the EDC activation is added among the DAH/EDC/AuNPs 4 ℃ of reaction 6h.Reactant obtains the AuNPs-Ab conjugate in centrifugal 30 min of 5,000 rpm.
The preparation of immunosensor: get MWCNTs-CS nano-complex that 10 μ L prepare and drip and be coated in cleaned GCE surface, then infrared drying with the PBS damping fluid flushing surface of pH=7.0, dries, and this modified electrode is denoted as MWCNTs-CS/GCE; Get GS-PEI-Au nano-complex that 10 μ L prepare and drip and be coated in the MWCNTs-CS/GCE surface, then infrared drying with the PBS damping fluid flushing surface of pH=7.0, dries, and is denoted as GS-PEI-AuMWCNTs-CS/GCE; Get 10 μ L AuNPs-Ab conjugates and drip and be coated on the GS-PEI-AuMWCNTs-CS/GCE, hatch 12h for 4 ℃; With leaving standstill 30min under the room temperature in the BSA solution of the above-mentioned electrode immersion 1% that assembles, with nonspecific binding site on the enclosed-electrode, dry under the room temperature, immunosensor completes, and is kept under 4 ℃ of conditions for subsequent use.⑸ be optimized from test end liquid pH, incubation temperature and the incubation time three aspects: experiment condition to prepared immunosensor, and the scope of pH is 4.0-8.0, and the scope of incubation time is 10-40min.
Configuration 0.1-1.0 * 10
4The agricultural chemicals carbofuran standard solution of ng/mL immerses the above-mentioned immunosensor for preparing respectively the carbofuran standard solution of variable concentrations, hatches at normal temperatures 25min, detect immunity before and after curent change obtain its working curve;
With immunosensor in the situation that the chaff interferences such as chlopyrifos, DDVP, 3-hydroxyl carbofuran, sevin exist the carbofuran of 100ng/mL tested, to detect its selectivity; Select 5 immunosensors that prepare to detect 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;
Multiple fruits and vegetables sample is thoroughly cleaned up, carry out the test of actual sample recovery of standard addition.
The detection method operating procedure that this kind current mode immunosensor detects residues of pesticides 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 (2)
1. the immunosensor preparation method of an and nano composite material directed fixing based on antibody is characterized in that: prepared a kind of novel GS-PEI-Au nano composite material, be used for improving water-soluble, electric conductivity and the stability of Graphene; Adopt the common modified electrode of synergy of MWCNTs-CS and two kinds of composite nano materials of GS-PEI-Au, strengthened significantly current-responsive, enlarged effective fixed-area of electrode, improved effective fixed amount of electrode surface antibody.
2. the immunosensor preparation method of a kind of and nano composite material directed fixing based on antibody as claimed in claim 1, it is characterized in that: glutathione will resist the directed covalency of monoclonal antibody of carbofuran agricultural chemicals to be fixed to nanometer gold size surface as the space arm, prepared a kind of novel AuNPs-Ab conjugate, realized that antibody fixes in the orientation on glass-carbon electrode surface, improved the detection performance of immunosensor.
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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 |
| CN103592437A (en) * | 2013-11-11 | 2014-02-19 | 山东理工大学 | Immunosensor based on modification of graphene-multiwalled carbon-nanogold size-chitosan |
| CN103754863A (en) * | 2013-12-26 | 2014-04-30 | 湖南大学 | Preparation method of gold-graphene composite nano material |
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| CN111721819A (en) * | 2020-06-12 | 2020-09-29 | 创蔚来(平潭)科技有限公司 | Electrochemical aptamer sensor for simultaneously detecting multiple disease markers |
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