CN103472219B - Method combining immune magnetic nanoparticles and SPR technique to detect deltamethrin - Google Patents
Method combining immune magnetic nanoparticles and SPR technique to detect deltamethrin Download PDFInfo
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- CN103472219B CN103472219B CN201310460621.3A CN201310460621A CN103472219B CN 103472219 B CN103472219 B CN 103472219B CN 201310460621 A CN201310460621 A CN 201310460621A CN 103472219 B CN103472219 B CN 103472219B
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Abstract
Provided is a method combing immune magnetic nanoparticles and an SPR technique to detect deltamethrin. The method comprises the steps of adding the immune magnetic nanoparticles into sample solution, to be tested, of the deltamethrin to enable the immune magnetic nanoparticles to generate specific binding with the deltamethrin, obtaining conjugate through applied magnetic field separation, and directly injecting the conjugate into the surface of an SPR chip modified by chitosan to detect the deltamethrin content in the sample solution to be detected. The immune magnetic nanoparticles are obtained through the steps of activating carboxyl on the surfaces of the magnetic nanoparticles with DC/NHS mixed liquor, adding monoclonal antibody solution of the deltamethrin, then adding cholamine to be sealed and using PBS buffer solution to achieve constant volume after applied magnetic field separation to obtain the immune magnetic nanopartiles. The method combines sample pretreatment with a detection technique to effectively improve detection flexibility and accuracy. Furthermore, the method is simple and quick to operate, high in selectivity, good in reproducibility, capable of directly detecting deltamethrin residues in agricultural products and important in actual application value. In addition, the recovery rate meets the requirement.
Description
Technical field
The present invention relates to the analysis of agricultural drugs and biosensor technology field, concrete relate to a kind of SPR immunosensor method of utilizing immune magnetic nanometer to detect decis residual in agricultural product.
Background technology
Decis is the highest one of virulence in current chrysanthemum ester insecticide.Decis belongs to poisoning malicious class, and skin contact can cause irritation, occurs red papule.When acute poisoning, the lighter has headache, dizziness, feels sick, vomiting, poor appetite, weak, and severe one also can occur that muscle bundle trembles and twitches.The detection method of deltamethrin residues mainly contains gas chromatography, high performance liquid chromatography, Chromatography/Mass Spectrometry coupling technique, enzyme-linked immuno assay etc. at present.Wherein red, orange, green, blue, yellow (ROGBY) instrument and equipment is valuable, and sample pre-treatments complexity, needs professional; And enzyme-linked immune analytic method relative complex, sensitivity is lower, consuming time, and cost is higher, and all needs complicated sample pre-treatments.Compared with above-mentioned detection method, surface plasma resonance (surface plasmon resonance, SPR) sensing technology be a kind of based on material refractive index change, modern detecting that can real-time dynamic monitoring, have quick, highly sensitive, consumption sample amount is few, without characteristics such as mark, chip are reusable, and simple to operate, but cannot directly detect with sensitivity the little molecule of deltamethrin pesticide.
Pesticide residue analysis is the analytical technology of trace components in complex mixture, and adopting suitable residues of pesticides method of purification is successfully to set up key accurate, quick, highly sensitive the Detection Technologies of Pesticide Residues.The magnetic Nano material of development in recent years is a kind of novel compatibility solid phase carrier, has the feature of controlled motion under externally-applied magnetic field, and equipment needed thereby is simple, operates soft.Magnetic nanometer is combined and is applied to detection with various analytical approachs, can be in complicated system fast enriching thing to be checked, concentrated, pure sample, contribute to shorten the accuracy of detection time and detection.
Summary of the invention
Technical matters to be solved by this invention is, cannot realize the little molecule of direct highly sensitive detection deltamethrin pesticide for existing spr sensor, and the problem of agricultural product sample pre-treatments complexity, provide a kind of and adopt immune magnetic nanometer and SPR technology to combine to detect the method for decis.The method not only combines sample pre-treatments and detection technique, sensitivity and the accuracy of method are improved, and simple to operate, detect fast, selectivity is high, reappearance and reproducibility good, the recovery meets the requirements, can directly detect deltamethrin residues in agricultural product, there is important actual application value.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: a kind ofly adopt immune magnetic nanometer and SPR technology to combine to detect the method for decis, the method is that immune magnetic nanometer is joined in the testing sample solution containing decis, make immune magnetic nanometer and decis generation specific binding, after separating, externally-applied magnetic field obtains bond, this bond is directly injected through chitosan-modified SPR chip surface, to detect the decis content in testing sample solution.
Details are as follows for the process of above-mentioned detection decis, incorporated by reference to referring to Fig. 1:
1, the Preparation and characterization of magnetic nanometer: get 0.5g fulvic acid and add 60mL pure water and be placed in round-bottomed flask, when passing into nitrogen deoxygenation, be also heated with stirring to boiling with constant temperature blender with magnetic force 200r/min, after boiling, add immediately 5mL to contain 0.6g NaOH, 1.08g FeCl
36H
2o and 0.6g FeSO
47H
2the mixed solution of O, backflow 2h.After solution is cooled to room temperature, through magnetic resolution, repeatedly pure water washing, to neutral, adopts respectively ultraviolet spectrum, Fourier's infrared spectrum and transmission electron microscope to characterize, in conjunction with referring to Fig. 2.
The preparation method of above-mentioned carboxylated magnetic nanometer is prior art, can be with reference to the synthetic method of the carboxylated magnetic nanometer in surface of having reported [Chunjiao Zhou et al, Fulvic acid coated iron oxide nanoparticles for magnetic resonance imaging contrast agent, Funct.Mater.Lett.03 (2010) 197-200 page].
2, the preparation of immunity magnetic nanometer: the carboxylated magnetic nanometer solution of getting 200 μ L1.9-3.8mM, add the EDC/NHS(0.4M/0.1M of 400 μ L) mixed liquid, whirlpool concussion 5-10min, after ice-bath ultrasonic 5-10min, add 200 μ L0.27-0.55mg/mL decis monoclonal antibody solutions, whirlpool concussion 5-10min, after ice-bath ultrasonic 5-10min, add 0.1M monoethanolamine 200 μ L, whirlpool concussion 5-10min, leave standstill after 15-20min, after externally-applied magnetic field separates, with PBS damping fluid (10mM, pH7.4) clean and be settled to 200 μ L, being placed in 4 DEG C of refrigerators saves backup, adopt respectively ultraviolet spectrum, Fourier's infrared spectrum and transmission electron microscope characterize, in conjunction with referring to Fig. 2.
The decis monoclonal antibody solution of above-mentioned 0.27-0.55mg/mL is to get 3.3mg decis monoclonal antibody (buying in Wuhan Sanying Bio-Technology Co., Ltd.) to add 1ml PBS damping fluid (10mM, pH7.4) to be mixed with the mother liquor of 3.3mg/mL, then uses PBS damping fluid (10mM, pH7.4) dilution to form.
3, the structure of spr sensor: SPR chip is soaked to 3-8min in absolute ethyl alcohol, through pure water rinsing, after nitrogen dries up, with hydrogen flame calcination 15-30s, after cooling, pack SPR instrument into, after baseline is walked to put down, pass into the mercaptoethylmaine solution of 50-100 μ L0.4mg/mL with the flow velocity of 10-20 μ L/min, pass into again the chitosan-acetic acid solution of 50-100 μ L1.2-1.8mg/mL with the flow velocity of 10-20 μ L/min, make this chitosan-acetic acid solution carry out self assembly at SPR chip surface and (utilize the amino of mercaptoethylmaine and the carboxyl reaction on shitosan surface, make shitosan be fixed on chip surface) make.
The chitosan-acetic acid solution of above-mentioned 1.2-1.8mg/mL is to get 200mg shitosan to be dissolved in 50mL50mM acetic acid-sodium-acetate buffer (pH3.6), 60 DEG C of magnetic agitation 2-4h, cooling after, then use acetic acid-sodium-acetate buffer (pH3.6) dilution of 50mM to form.
4, immune magnetic nanometer efficiently concentrating determinand decis: the immune magnetic nanometer of getting above-mentioned preparation adds the sample solution that contains decis, after whirlpool concussion 5-10min, ice-bath ultrasonic 5-10min, then leave standstill 15-20min, after separating, externally-applied magnetic field obtains bond, PBS damping fluid (10mM, pH7.4) cyclic washing 3 times of this bond, with for subsequent use after PBS constant volume.
5, the bond of immune magnetic nanometer obtained above and decis is directly injected to sensor and detects, can obtain decis in sample solution content.
The present invention utilizes immune magnetic nanometer and decis that specific binding can occur, the bond obtaining after externally-applied magnetic field separates directly injects through chitosan-modified SPR chip surface, can there is Huffman alkylated reaction in the amino on shitosan surface and the bromine atoms of decis, the refractive index of chip surface is changed along with the variation of decis concentration in bond, thereby cause the variation of SPR response signal, can directly carry out quantitative test to decis accordingly, lowest detection is limited to 2.5pg/mL.
The present invention and traditional detection method and same type of sensor comparison, the detectability of its 2.5pg/mL is lower than most of bibliographical informations, in table 1.
The contrast of table 1 the present invention and traditional detection method and same type of sensor
Compared with prior art, advantage of the present invention is as follows:
1, the present invention utilizes the specific surface area of magnetic nanometer to make it fixing antibody amount increases, and immune magnetic nanometer itself has large refractive index, effectively improved the response signal of SPR, made the new spr sensor building have very high sensitivity, lowest detection is limited to 2.5pg/mL.
2, the present invention utilizes immune magnetic nanometer and decis generation specific binding, the bond obtaining after externally-applied magnetic field separates directly carries out SPR detection, and Sample Pretreatment Technique is combined with detection technique, has effectively improved the accuracy detecting, and simple to operate, detection speed is fast.In addition this spr sensor has good specificity, reappearance and the recovery, can directly detect deltamethrin residues in agricultural product, has important actual application value.
Brief description of the drawings
Fig. 1 is that decis sensor of the present invention builds schematic diagram.
Wherein, a represents magnetic nanometer, and b represents monoethanolamine, and c represents the monoclonal antibody of decis, and d represents decis, and e represents shitosan, and f represents mercaptoethylmaine, and g represents golden film, and h represents slide, and i represents prism.
Fig. 2 is the ultraviolet phenogram of magnetic nanometer in the present invention, immune magnetic nanometer and specific binding decis compound thereof.
Wherein: a represents the monoclonal antibody+decis+shitosan of magnetic nanometer+decis, b represents the monoclonal antibody of magnetic nanometer+decis, c represents the monoclonal antibody of decis+decis, d represents magnetic nanometer, e represents decis, f represents the monoclonal antibody of decis, and g represents shitosan+mercaptoethylmaine.
Fig. 3 is the specificity of spr sensor of the present invention.
Wherein, a represents the mixed liquor of decis, Atrazine and fenvalerate, and b represents the mixed liquor of decis and fenvalerate, and c represents the mixed liquor of decis and Atrazine, and d represents decis, and e represents Atrazine, and f represents fenvalerate.
Fig. 4 is the SPR kinetic curve that the present invention detects decis.
Wherein, the decis concentration of each curve representative is respectively: a--25ng/mL, b--10ng/mL, c--5ng/mL, d--1ng/mL, e-750pg/mL, f--500pg/mL, g--250pg/mL, h--100pg/mL, i--50pg/mL, j--25pg/mL, k--10pg/mL.
Fig. 5 is the typical curve that the present invention detects decis.
Embodiment:
The specificity experiment of the spr sensor that embodiment 1, the present invention build
The SPR immunosensor that the present invention is built is the fenvalerate, the Atrazine of 1ng/mL of the decis to 1ng/mL, 1ng/mL respectively, the potpourri of the Atrazine of the decis of 1ng/mL and the fenvalerate of 1ng/mL and 1ng/mL, the potpourri of the decis of 1ng/mL and the fenvalerate of 1ng/mL, and the potpourri of the decis of 1ng/mL and the Atrazine of 1ng/mL detects, the variation of more corresponding SPR response signal, investigates the specificity of this sensor.Result shows, in conjunction with referring to Fig. 3, the spr sensor that the present invention builds has good specificity.
The foundation of embodiment 2, detection decis typical curve
Be under the condition of 10 μ L/min at flow velocity by the spr sensor building (concentration of chitosan-acetic acid solution is 1.5mg/mL), the bond (0,0.010 of the variable concentrations decis to immune magnetic nanometer specific binding, 0.025,0.050,0.075,0.1,0.5,0.25ng/mL, n=3) detect, set up the typical curve that detects decis, the range of linearity: 0.01-1ng/mL, lowest detection is limited to 2.5pg/mL, in conjunction with referring to Fig. 4 and Fig. 5.
Embodiment 3, recovery of standard addition experiment
Get appropriate decis standard specimen and add in soybean sample, arrange 0.75,0.5, tri-concentration of 0.25ng/mL, each concentration duplicate detection three times, investigates the accuracy of the spr sensor of this structure.
The pre-treatment of soybean sample: pulverize and cross 100 mesh sieves after soybean drying, claim 1g soy meal to be added in 6mL80% methanol solution, after concussion 10min, with the centrifugal 3min of 10000rpm.Get supernatant, and become 0.1g/mIJ solution by 80% methanol constant volume, then after 0.2p m film is crossed film and processed, be diluted to 4 DEG C of preservations of 1mg/mL solution, if soybean sample is liquid, directly add decis standard specimen.
Testing result is in table 2, and the soybean recovery is 96.94-103.60%, the coefficient of variation 2.18-13.72%, and the recovery, in allowed band (low concentration recovery allowed band is 70-130%), meets the requirement of the recovery.
The experiment of table 2 recovery
Claims (4)
1. one kind adopts immune magnetic nanometer and SPR technology to combine to detect the method for decis, it is characterized in that, the method is that immune magnetic nanometer is joined in the testing sample solution containing decis, make immune magnetic nanometer and decis generation specific binding, after separating, externally-applied magnetic field obtains the bond of immune magnetic nanometer and decis, this bond is directly injected through chitosan-modified SPR chip surface, obtain SPR response signal, to detect the decis content in testing sample solution;
Wherein, above-mentioned immune magnetic nanometer is the carboxylated magnetic nanometer solution of getting 200 μ L1.9-3.8mM, add the EDC of 0.4M and the NHS mixed solution of 0.1M totally 400 μ L, whirlpool concussion 5-10min, after ice-bath ultrasonic 5-10min, add again 200 μ L0.27-0.55mg/mL decis monoclonal antibody solutions, whirlpool concussion 5-10min, ice-bath ultrasonic 5-10min, adds 0.1M monoethanolamine 200 μ L, whirlpool concussion 5-10min, leave standstill 15-20min, after externally-applied magnetic field separates, by PBS buffer solution for cleaning and be settled to 200 μ L, obtain.
2. the immune magnetic nanometer of employing as claimed in claim 1 and SPR technology combine and detect the method for decis, it is characterized in that, described decis monoclonal antibody solution is to get 3.3mg decis monoclonal antibody to add PBS damping fluid 1ml to be mixed with the mother liquor of 3.3mg/mL, then dilutes and form with PBS damping fluid.
3. the immune magnetic nanometer of employing as claimed in claim 1 and SPR technology combine and detect the method for decis, it is characterized in that, described is that SPR chip is soaked to 3-8min in absolute ethyl alcohol through chitosan-modified SPR chip, through pure water rinsing, after nitrogen dries up, with hydrogen flame calcination 15-30s, after cooling, pack SPR instrument into, after baseline is walked to put down, pass into the mercaptoethylmaine solution of 50-100 μ L0.4mg/mL with the flow velocity of 10-20 μ L/min, pass into again the chitosan-acetic acid solution of 50-100 μ L1.2-1.8mg/mL with the flow velocity of 10-20 μ L/min, making this chitosan-acetic acid solution carry out self assembly at SPR chip surface makes.
4. the immune magnetic nanometer of employing as claimed in claim 3 and SPR technology combine and detect the method for decis, it is characterized in that, described chitosan-acetic acid solution is to get 200mg shitosan to be dissolved in pH value be in 50mL50mM acetic acid-sodium-acetate buffer of 3.6,60 DEG C of magnetic agitation 2-4h, after cooling, then the dilution of the acetic acid of the 50mM that is 3.6 by pH value-sodium-acetate buffer forms.
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