CN103472219A - 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
- Publication number
- CN103472219A CN103472219A CN2013104606213A CN201310460621A CN103472219A CN 103472219 A CN103472219 A CN 103472219A CN 2013104606213 A CN2013104606213 A CN 2013104606213A CN 201310460621 A CN201310460621 A CN 201310460621A CN 103472219 A CN103472219 A CN 103472219A
- Authority
- CN
- China
- Prior art keywords
- decis
- spr
- deltamethrin
- solution
- detect
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
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 a kind of of virulence in current chrysanthemum ester insecticide.Decis belongs to poisoning malicious class, and skin contact can cause irritation, red papule occurs.During 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 the red, orange, green, blue, yellow (ROGBY) instrument and equipment is valuable, and the sample pre-treatments complexity, need the professional; And the enzyme-linked immune analytic method relative complex, sensitivity is lower, consuming time, and cost is higher, and all needs complicated sample pre-treatments.With above-mentioned detection method, compare, surface plasma resonance (surface plasmon resonance, SPR) but sensing technology is a kind of modern detecting that changes real-time dynamic monitoring based on material refractive index, have quick, highly sensitive, consumption sample amount is few, without characteristics such as mark, chip are reusable, and simple to operate, but can't 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 characteristics of controlled motion under externally-applied magnetic field, and equipment needed thereby is simple, operates soft.Magnetic nanometer is combined with various analytical approachs and is applied to detect, 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, can't 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, a kind of method that adopts immune magnetic nanometer and SPR technology to combine to detect decis is provided.The method not only combines sample pre-treatments and detection technique, sensitivity and the accuracy of method have been improved, and simple to operate, detect fast, selectivity is high, reappearance and reproducibility good, the recovery meets the requirements, can directly to deltamethrin residues in agricultural product, be detected, be 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 of method that adopts immune magnetic nanometer and SPR technology to combine to detect 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, please in conjunction with referring to Fig. 1:
1, the Preparation and characterization of magnetic nanometer: get the 0.5g fulvic acid and add the 60mL pure water and be placed in round-bottomed flask, also with constant temperature blender with magnetic force 200r/min, be heated with stirring to boiling when passing into the nitrogen deoxygenation, 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 synthetic method [the Chunjiao Zhou et al of the carboxylated magnetic nanometer in surface of having reported, 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, the EDC/NHS(0.4M/0.1M that adds 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, after standing 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 ℃ of refrigerators saves backup, adopt respectively ultraviolet spectrum, Fourier's infrared spectrum and transmission electron microscope are characterized, 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: the 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, the SPR instrument of packing into after cooling, 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 the 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 the 200mg shitosan to be dissolved in 50mL50mM acetic acid-sodium-acetate buffer (pH3.6), 60 ℃ 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 standing 15-20min, after separating, externally-applied magnetic field obtains bond, PBS damping fluid (10mM, pH7.4) cyclic washing 3 times of this bond, with standby after the PBS constant volume.
5, the bond of immune magnetic nanometer obtained above and decis is directly injected to sensor and is detected, can obtain decis in sample solution content.
The present invention utilizes immune magnetic nanometer and decis that specific binding can occur, the bond obtained after externally-applied magnetic field separates directly injects through chitosan-modified SPR chip surface, the Huffman alkylated reaction can occur 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.
Relatively, the detectability of its 2.5pg/mL is lower than most of bibliographical informations, in Table 1 for the present invention and traditional detection method and same type of sensor.
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 spr sensor of new structure 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 obtained after externally-applied magnetic field separates directly carries out the SPR detection, and Sample Pretreatment Technique is combined with detection technique, has effectively improved the accuracy detected, and simple to operate, detection speed is fast.This spr sensor has good specificity, reappearance and the recovery in addition, can directly to deltamethrin residues in agricultural product, be detected, and has important actual application value.
The accompanying drawing explanation
Fig. 1 is that decis sensor of the present invention builds schematic diagram.
Wherein, a means magnetic nanometer, and b means monoethanolamine, and c means the monoclonal antibody of decis, and d means decis, and e means shitosan, and f means mercaptoethylmaine, and g means golden film, and h means slide, and i means prism.
The ultraviolet phenogram that Fig. 2 is magnetic nanometer in the present invention, immune magnetic nanometer and specific binding decis compound thereof.
Wherein: a means the monoclonal antibody+decis of magnetic nanometer+decis+shitosan, b means the monoclonal antibody of magnetic nanometer+decis, c means the monoclonal antibody of decis+decis, d means magnetic nanometer, e means decis, f means the monoclonal antibody of decis, and g means shitosan+mercaptoethylmaine.
The specificity that Fig. 3 is spr sensor of the present invention.
Wherein, a means the mixed liquor of decis, Atrazine and fenvalerate, and b means the mixed liquor of decis and fenvalerate, and c means the mixed liquor of decis and Atrazine, and d means decis, and e means Atrazine, and f means 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 respectively to the decis of 1ng/mL, the fenvalerate of 1ng/mL, the Atrazine of 1ng/mL, 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 Atrazine of the decis of 1ng/mL and 1ng/mL is detected, the variation of more corresponding SPR response signal, investigate 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
Under the condition that is 10 μ L/min at flow velocity by the spr sensor (concentration of chitosan-acetic acid solution is 1.5mg/mL) that builds, to the bond (0,0.010 of the variable concentrations decis of immune magnetic nanometer specific binding, 0.025,0.050,0.075,0.1,0.5,0.25ng/mL, n=3) detected, 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, investigate the accuracy of the spr sensor of this structure.
The pre-treatment of soybean sample: pulverize and cross 100 mesh sieves after the soybean drying, claim the 1g soy meal to be added in the 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 the film processing, be diluted to 4 ℃ of preservations of 1mg/mL solution, as soybean sample is liquid, directly add the 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 (5)
1. a method that adopts immune magnetic nanometer and SPR technology to combine to detect 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 bond, this bond is directly injected through chitosan-modified SPR chip surface, to detect the decis content in testing sample solution.
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 immune magnetic nanometer is the carboxylated magnetic nanometer solution of getting 200 μ L1.9-3.8mM, the NHS mixed solution that adds the EDC of 0.4M and 0.1M is 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, add 0.1M monoethanolamine 200 μ L, whirlpool concussion 5-10min, standing 15-20min, after externally-applied magnetic field separates, by the PBS buffer solution for cleaning and be settled to 200 μ L, obtain.
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 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 forms with the dilution of PBS damping fluid.
4. 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 the 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, the SPR instrument of packing into after cooling, 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 the SPR chip surface makes.
5. chitosan-modified SPR chip as claimed in claim 4, it is characterized in that, described chitosan-acetic acid solution is to get the 200mg shitosan to be dissolved in 50mL50mM acetic acid-sodium-acetate buffer that the pH value is 3.6,60 ℃ of magnetic agitation 2-4h, after cooling, then the acetic acid of the 50mM that is 3.6 by the pH value-sodium-acetate buffer dilution forms.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310460621.3A CN103472219B (en) | 2013-09-30 | 2013-09-30 | Method combining immune magnetic nanoparticles and SPR technique to detect deltamethrin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310460621.3A CN103472219B (en) | 2013-09-30 | 2013-09-30 | Method combining immune magnetic nanoparticles and SPR technique to detect deltamethrin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103472219A true CN103472219A (en) | 2013-12-25 |
| CN103472219B CN103472219B (en) | 2014-06-11 |
Family
ID=49797146
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310460621.3A Active CN103472219B (en) | 2013-09-30 | 2013-09-30 | Method combining immune magnetic nanoparticles and SPR technique to detect deltamethrin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103472219B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108139398A (en) * | 2015-10-23 | 2018-06-08 | 阿莫生命科学有限公司 | The protein fixing means of particle |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101629909A (en) * | 2008-07-20 | 2010-01-20 | 欧普图斯科技有限公司 | Method for detecting chemical or biological substance |
| CN102072894A (en) * | 2009-11-25 | 2011-05-25 | 欧普图斯(苏州)光学纳米科技有限公司 | Nano-structure-based spectrum detecting method for detecting chemical and biochemical impurities |
| CN102519912A (en) * | 2011-12-06 | 2012-06-27 | 深圳市检验检疫科学研究院 | Method for detecting object to be detected by using surface plasmon resonance (SPR) biosensor |
| CN102628803A (en) * | 2012-04-17 | 2012-08-08 | 王利兵 | Method for detecting pesticide and veterinary medicament residues in foods based on surface plasma resonance technology |
| CN102947703A (en) * | 2010-06-17 | 2013-02-27 | 皇家飞利浦电子股份有限公司 | Multi epitope assay |
-
2013
- 2013-09-30 CN CN201310460621.3A patent/CN103472219B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101629909A (en) * | 2008-07-20 | 2010-01-20 | 欧普图斯科技有限公司 | Method for detecting chemical or biological substance |
| CN102072894A (en) * | 2009-11-25 | 2011-05-25 | 欧普图斯(苏州)光学纳米科技有限公司 | Nano-structure-based spectrum detecting method for detecting chemical and biochemical impurities |
| CN102947703A (en) * | 2010-06-17 | 2013-02-27 | 皇家飞利浦电子股份有限公司 | Multi epitope assay |
| CN102519912A (en) * | 2011-12-06 | 2012-06-27 | 深圳市检验检疫科学研究院 | Method for detecting object to be detected by using surface plasmon resonance (SPR) biosensor |
| CN102628803A (en) * | 2012-04-17 | 2012-08-08 | 王利兵 | Method for detecting pesticide and veterinary medicament residues in foods based on surface plasma resonance technology |
Non-Patent Citations (7)
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108139398A (en) * | 2015-10-23 | 2018-06-08 | 阿莫生命科学有限公司 | The protein fixing means of particle |
| US11169147B2 (en) | 2015-10-23 | 2021-11-09 | Amolifescience Co., Ltd. | Method for immobilizing protein on particle |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103472219B (en) | 2014-06-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104592446B (en) | The preparation method and application of the nanofiber molecularly imprinted polymer of tetracycline antibiotics | |
| CN108828208A (en) | A kind of aflatoxin pretreatment reagent kit using immunomagnetic bead technique | |
| CN109991419A (en) | Nicotinic insecticide thiacloprid method for detecting residue based on SPR technique | |
| CN101158685A (en) | Producing and use method of vomitus toxin semi-fix quantify speed testing agent strip | |
| CN103472219B (en) | Method combining immune magnetic nanoparticles and SPR technique to detect deltamethrin | |
| CN104034886A (en) | Immune colloidal gold test strip for detecting organic phosphorus pesticide and preparation method thereof | |
| CN108982842A (en) | A kind of zearalenone pretreatment reagent kit using immunomagnetic bead technique | |
| CN105319354A (en) | Preparation method of chemiluminescence immunoassay detection kit used for detecting methyl parathion | |
| CN104910339A (en) | Magnetic molecular imprinting polyion liquid for detecting ractopamine as well as preparation method and application of magnetic molecular imprinting polyion liquid | |
| CN103048476A (en) | Thyroxine nanometer magnetic particle chemiluminiscence determinstion kit as well as preparation method and detection method thereof | |
| CN109061198A (en) | inhibin A detection kit and preparation method thereof | |
| Xu et al. | Simultaneous detection of diethylstilbestrol and estradiol residues with a single immunochromatographic assay strip | |
| CN101701960B (en) | Preparation method of conjugate for detecting microcystin | |
| CN103983613B (en) | Ractopamine molecular engram spr sensor chip and preparation method thereof | |
| CN108918850A (en) | The quickly method of the aflatoxin in detection medicinal material | |
| CN106350576B (en) | A method of clenobuterol hydrochloride residue is detected based on blood glucose meter | |
| CN104017774A (en) | Anti-chlorothalonil monoclonal antibody hybridoma cell strain and application thereof | |
| CN103969431B (en) | A kind of preparation method of the immunomagnetic beads for hidden state T-2 toxin enrichment purification | |
| CN208224280U (en) | A kind of quickly detection pure and mild chloramphenicol Immunofluorescence test paper strip of Gibberella zeae | |
| CN105112376A (en) | Amantadine monoclonal antibody hybridoma cell strain and application thereof | |
| CN103145633B (en) | Novel melamine antigen and antibody and application | |
| CN103901200A (en) | Magnetic immunochromatographic test strip for detecting tobacco mosaic virus (TMV) and preparation method thereof | |
| CN102507940B (en) | Method for quickly detecting chloramphenicol in cosmetics | |
| CN101482565A (en) | Melamine colloidal gold immunochromatography detection test paper and its production method | |
| CN202649164U (en) | Electrochemical modified electrode used for detecting clenbuterol and detector |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |