CN102879575B - 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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- CN102879575B CN102879575B CN201210405208.2A CN201210405208A CN102879575B CN 102879575 B CN102879575 B CN 102879575B CN 201210405208 A CN201210405208 A CN 201210405208A CN 102879575 B CN102879575 B CN 102879575B
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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 immunosensor preparation method who the invention provides a kind of and nano composite material directed fixing based on antibody, belongs to 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 fruits and vegetables is the focus that people pay close attention to always, and after being also China's entry into the WTO now, 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 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 forbidden in the world in recent years this agricultural chemicals on vegetables, because it has growth stimulation to some crop, so still have many phenomenons that use violating the regulations.There are the fruits and vegetables that highly toxic like this residues of pesticides exceed standard and come into the market, will cause serious harm to health of people, therefore set up a kind of quick, sensitive, safe and reliable Detecting Pesticide analytical approach 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 met.Immunosensor is the sensor that the immunoreactive principle that causes according to antigen and antibody specific binding is developed into, and compared with traditional analytical approach, 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) 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.
Summary of the invention
The object of invention is to provide one can overcome above-mentioned defect, 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 antibody directed fixing, the monoclonal antibody (Ab) of anti-carbofuran is directed covalency and is fixed to nanometer gold size surface using glutathione (glutathione, G-SH) as space arm.
The immunosensor preparation method of described a kind of fixing based on antibody orientation and nano composite material, 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 fixing based on antibody orientation and nano composite material, is characterized in that: the optimization of experiment condition, mainly comprises pH, incubation temperature and the incubation time of testing 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; Immunosensor Performance Detection comprises specificity, stability, reappearance, reproducibility and the immunosensor mensuration to multiple fruits and vegetables sample recovery rate.
Its preparation principle is: the present invention adopts glutathione (glutathione, G-SH) as space arm, the directed covalency of the monoclonal antibody of anti-carbofuran (Ab) is fixed to nanometer gold size surface, wherein G-SH is firmly 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, is connected to nanometer gold size surface thereby realized Ab orientation.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), strengthen significantly current-responsive, expand effective fixed-area of electrode, improved effective fixed amount of electrode surface antibody.To make after immunosensor through the electrode of above-mentioned steps modified, detect the precision of agricultural chemicals higher, realize object small-sized, convenient, that be applicable to Site Detection.Before the immunosensor that adopts the present invention to make can be gathered at vegetables and fruits, be gone on the market, carry out the Fast Measurement of agricultural chemicals Determination of carbofuran, directly to residues of pesticides, whether superscale detects, avoid because of the vegetables and fruits that food contains remains of pesticide cause poisoning, for crop production safety and consumption provide the technical support of residue detection.
For reaching above object, 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: (1) by covalent approach by 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; (2) the synergy based on MWCNTs-CS and two kinds of nano composite materials of GS-PEI-Au, by its common modified electrode.
The present invention has prepared a kind of novel nano-material GS-PEI-Au nano composite material, 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, increase the useful area of electrode for fixing more antibody.In addition, fixedly keep the biologically active of antibody and antigen binding site freely by the orientation of antibody, strengthened the detection sensitivity of sensor.
The preparation technology of described current mode immunosensor is as follows: (1) prepare MWCNTs-CS nano composite material, GS-PEI-Au nano composite material and AuNPs-Ab conjugate;
mWCNTs-CS nano composite material is modified to cleaned naked glass-carbon electrode and modify above, dry;
modify GS-PEI-Au nano composite material, dry;
modify AuNPs-Ab conjugate, hatch 12h for 4 ℃; (6) by leaving standstill 30min under room temperature in the BSA solution of the last immersion 1% of the above-mentioned electrode preparing, with nonspecific binding site on enclosed-electrode, under room temperature, dry, immunosensor completes, and is kept under 4 ℃ of conditions for subsequent use.
Embodiment
Embodiment: the (1) preparation of MWCNTs-CS nano composite material: shitosan (CS) is dissolved in 1.0% acetum, is made into 0.25% CS solution, room temperature lower magnetic force stirs 1h, and shitosan is dissolved completely, then with NaOH, the pH of mixed liquor is transferred to 5.0.Take 1mg MWNTs, add 1mL 0.25% chitosan solution, more ultrasonic dispersion 2h, stable black dispersion liquid (MWCNTs-CS nano composite material) finally obtained.
the preparation of GS-PEI-Au nano composite material: take 3mg GS, add 10mL deionized water, ultrasonic dispersion 10min, obtains GS dispersion liquid.In GS dispersion liquid, add 0.2 mL 1.0 M PEI aqueous solution, ultrasonic dispersion 10min, then adds 1.6 mL 21mM HAuCl
4, by 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, repeatedly centrifugal, and centrifugal gained sediment is dissolved in 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 the stability of one month.
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 in 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), in a cantilever arrangement, at 4 ℃, place 1.5 h, then in 15, under 000 rpm, centrifugal 1 h isolates the AuNPs(EDC/AuNPs that EDC has activated), this reactant is suspended in 1 mL PBS.In this reactant, add 50 μ L 500 μ M DAH, under room temperature, mix 30min, the reaction mixture of gained is again centrifugal, obtains the AuNPs (DAH/EDC/AuNPs) that DAH has activated, and is suspended in 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, in a cantilever arrangement, at 4 ℃, place 1.5 h.Ab after EDC activation is added in DAH/EDC/AuNPs to 4 ℃ of reaction 6h.Reactant, in centrifugal 30 min of 5,000 rpm, obtains AuNPs-Ab conjugate.
the preparation of immunosensor: get MWCNTs-CS nano-complex that 10 μ L prepare and drip and be coated in cleaned GCE surface, infrared drying, then rinses surface with the PBS damping fluid 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 MWCNTs-CS/GCE surface, infrared drying, then rinses surface with the PBS damping fluid of pH=7.0, dries, and is denoted as GS-PEI-AuMWCNTs-CS/GCE; Get 10 μ L AuNPs-Ab conjugates and drip that to be coated in GS-PEI-AuMWCNTs-CS/GCE upper, hatch 12h for 4 ℃; By leaving standstill 30min under room temperature in the BSA solution of the above-mentioned electrode immersion 1% assembling, with nonspecific binding site on enclosed-electrode, under room temperature, dry, immunosensor completes, and is kept under 4 ℃ of conditions for subsequent use.(5) from test end liquid pH, incubation temperature and incubation time three aspects:, the experiment condition of prepared immunosensor is optimized, 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 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;
immunosensor in the situation that existing, is tested the carbofuran of 100ng/mL the chaff interferences such as chlopyrifos, DDVP, 3-hydroxyl carbofuran, sevin, to detect its selectivity; Select 5 immunosensors that prepare to detect its reappearance; The continuous carbofuran solution that detects successively same concentrations for 7 days is to detect its stability; By the immunity again after carbofuran agricultural chemicals 5min of dissociating of glycocoll-HCl damping fluid (pH 2.8) for the sensor after immunity, detect 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 of this kind of current mode immunosensor detection residues of pesticides is simple, detection time is shorter, 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 (1)
1. an immunosensor preparation method for and nano composite material directed fixing based on antibody, is characterized in that: prepared a kind of GS-PEI-Au nano composite material, for improving water-soluble, electric conductivity and the stability of Graphene; The common modified electrode of synergy that adopts MWCNTs-CS and two kinds of composite nano materials of GS-PEI-Au, has strengthened current-responsive significantly, has expanded effective fixed-area of electrode, has improved effective fixed amount of electrode surface antibody; Glutathione is fixed to nanometer gold size surface as space arm by directed the monoclonal antibody of anti-carbofuran agricultural chemicals covalency, prepare a kind of AuNPs-Ab conjugate, realize antibody and fixed in the orientation on glass-carbon electrode surface, improved the detection performance of immunosensor.
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| CN103592437B (en) * | 2013-11-11 | 2015-05-13 | 山东理工大学 | Immunosensor based on modification of graphene-multiwalled carbon-nanogold size-chitosan |
| CN103754863B (en) * | 2013-12-26 | 2015-04-22 | 湖南大学 | Preparation method of gold-graphene composite nano material |
| CN106680502A (en) * | 2016-06-17 | 2017-05-17 | 山东理工大学 | Preparation method and application of PSA (Prostate-specific Antigen) sensor based on silver shell encapsulated L-cysteine capture gold core label |
| CN109916981B (en) * | 2019-03-07 | 2021-03-02 | 江西农业大学 | Preparation method of double-signal electrochemical sensor for rapidly and selectively detecting sunset yellow |
| CN111721819B (en) * | 2020-06-12 | 2022-10-18 | 创蔚来(平潭)科技有限公司 | Electrochemical aptamer sensor for simultaneously detecting multiple disease markers |
| CN113899898B (en) * | 2021-10-15 | 2023-12-19 | 江苏省农业科学院 | Carbofuran immunodetection method and antibody based on reverse fluorescence enhanced double-channel signal |
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