CN109991419A - Nicotinic insecticide thiacloprid method for detecting residue based on SPR technique - Google Patents
Nicotinic insecticide thiacloprid method for detecting residue based on SPR technique Download PDFInfo
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Abstract
The invention discloses one kind for detecting the remaining SPR immune sensing detection method of anabasine insecticide thiacloprid.The present invention is using antigen-antibody immune response as basic principle, using antibody as sensor sensing recognition component, using direct competition method quickly, facilitate in conjunction with SPR technique to insecticide thiacloprid carry out residue detection, thiacloprid monoclonal antibody is fixed on SPR chip surface by chemical bond, cause signal in conjunction with the thiacloprid small molecule in prepare liquid, its detection sensitivity is improved, detection orientation is improved, the indirect competition for establishing signal amplification inhibits method.The minimum detectability of method is 0.197ng/mL, IC50For 2.13ng/mL, detection range is between 1.18-2.81ng/mL, so that detection is sensitiveer.
Description
Technical field
The invention belongs to Detecting Pesticide fields, are related to a kind of anabasine insecticide thiacloprid based on SPR technique
Detection method.
Background technique
Global warming, pest and disease damage is high-incidence, and to ensure that agricultural product high yield increases income, every kind of crop has to apply from harvest is seeded into
With Multiple Pesticides perhaps, more there is part peasant blindness pursuit picking time in advance and market appearance, fail reasonable standard using pesticide, lead
The residual exceeded event frequency of agriculture is caused to have generation.Especially anabasine insecticide is widely used, and mainly prevents and treats crop are as follows: vegetables,
The operatic circle and drupe, citrus, rice, cotton, corn, potato, sugar beet, rape and soybean etc..Residue problem is also very serious,
Especially economic well-being of workers and staff is higher, on fresh-keeping difficult, instant kind such as strawberry, red bayberry berry, Pesticide Residue is deposited
In no small risk hidden danger.Anabasine pesticide has the characteristics of efficient, less toxic and wide spectrum, as it is applied increasingly in the whole world
Extensively, health risk caused by it being widely used also more and more attention has been paid to.
Thiacloprid (Thiacloprid, cas:111988-49-9), chemical name (3- ((6- chloro-3-pyridyl base) methyl) -1,
3- thiazoline -2- subunit) cyanamide, molecular formula: C10H9ClN4S, molecular weight: 252.72, it is the novel chlorine developed by Bayer agrochemical
For nicotinic insecticide, there is stronger interior suction, tag and stomach poison function, be the height for preventing and treating pierce-suck type and pests with chewing mouthparts
Imitate medicament.2013, European Union raised difficult questions first to Diacloden, imidacloprid and clothianidin, has made and has limited the use of 2 years management measures, thiophene
Worm piperazine, imidacloprid, clothianidin, dinotefuran and Nitenpyram etc. are to honeybee high poison, and thiacloprid is less toxic to honeybee, this makes thiophene worm
Quinoline becomes leading role, dosage substantial increase from supporting role.Therefore, the pesticide residue of thiacloprid in environment and agricultural product is detected with non-
Often important meaning.
The method for detecting residue of thiacloprid at present mainly uses instrumental method, such as liquid phase chromatographic analysis method, liquid matter to join
With (HPLC-MS/MS) analytic approach etc., gas chromatography-mass spectrography method, molecular engram solid phase extraction-liquid chromatogram combination side
Method, ultrasonic wave added solid-liquid consolidate dispersion extraction-LC-MS method, dispersive liquid-liquid microextraction-LC-MS method, Capillary Electrophoresis
The analysis methods such as method.Although instrument analytical method high sensitivity, accuracy are strong, detecting instrument is expensive, sample pre-treatments are numerous
It is trivial, detection it is time-consuming and laborious, be difficult to meet the needs of the fast and convenient detection of a large amount of samples.
Although existing chemical method, chromatography, spectroscopic methodology, spectroscopic methodology, immunization, capillary electrophoresis and ion mobility spectrometry
The methods of use in Detecting Pesticide, but chemical method sensitivity is low, before chromatography, capillary electrophoresis and ion mobility spectrometry
The treatment process time is longer, needs dedicated technician and extensive expensive instrument, spectroscopic methodology sensitivity is low and treats test sample
Product purity requirement is more demanding, and the immunization operating time is too long.
Based on the biosensor of surface plasma resonance (Surface Plasmon Resonance, SPR) technology, it is
Point organically combined with bioactive materials (enzyme, protein, DNA, antibody, antigen, biomembrane etc.) and physical chemistry energy converter
Analyzer device is the quick and micro-analysis method of a kind of more advanced detection method and monitoring method and material molecule level.
Wherein SPR technique is the sensor technology based on affinity interaction.SPR technique has without label, to surface characteristic and object
Qualitative changeization is sensitive, in real time, quickly and easily realizes the features such as automation, is widely used in grinding for various interactions of molecules
Study carefully, such as the fields such as life science, clinical diagnosis, drug screening, food safety, environmental monitoring, monitoring object includes albumen, core
Acid, hormone, toxin, microorganism etc..Wherein detection of veterinary drugs in food, the various tumour micromolecular inhibitors of medicine screening in apply
It is more and more.But the application in pesticide research subject is fewer.
Biosensor based on surface plasma resonance (Surface Plasmon Resonance, SPR) effect is immune
Method is the new detection method of one kind developed in recent years.Surface plasma resonance sensor is a kind of multi-functional automatic point
Analyzer device can be monitored in real time intermolecular interaction and not need to mark.This method may be implemented multiple samples quickly,
The purpose accurately detected, after being detected due to each round, the reactant being incorporated on vane can be obtained with elution
To regeneration, vane is reused, repeats to detect, consumptive material is saved, is the frontier nature technology in food safety detection.
Its application is also extensive, can detecte vitamin, pesticide residue, biotoxin detection, bacterium and detection of pathogens.
Conventional antibodies still have certain limitation using upper immunization method, as polyclonal antibody still has homogeneity
It is poor, type is complicated, especially immune animal puts to death after taking blood and needs the problems such as being immunized again；Monoclonal antibody, as having
One of the natural antibody of classical immunoglobulin Y type structure, play a significant role (example in small molecule compound analysis detection
Such as, the pollutant in pesticide, mycotoxin, antibiotic and other environment and food).But small molecule is not because having immunogene
Property prepares antibody and generally requires to be coupled to the immune response that could excite animal after macromolecular carrier albumen, and conventional method is often
Need longer immune period (more than 4 times).There are a Research statistics, in the nearly 8700 fused cell holes of 10 groups of test for fusion, only 3
Strain of hybridoma can produce the antibody of identification oxytetracycline (antibiotic, MW=460.4), and positive rate is less than 0.1%.Cause
This, the preparation difficulty of small molecule compound antibody is higher than the preparation of macromolecular antigen-antibody.
It is reported in patent 201310460621.3 and detection decis is combined using immune nano particle and SPR technique
Method, but the preparation of immune nano particle needs first impregnated in dehydrated alcohol with SPR chip, pure water rinsing, nitrogen are blown
It is packed into SPR instrument after dry, hydrogen flame calcination, cooling, then flows into mercaptoethylmaine, then be passed through chitosan-acetic acid solution, makes chitosan
Acetum carries out self assembly in SPR chip surface and is made, and is then coupled the monoclonal of upper decis with EDC/NHS activation again
Antibody can just obtain immune nano particle, subsequent further to add small molecule decis sample to be measured, step on SPR instrument
Super more, process is cumbersome, although sensitivity is higher, does not have conveniently characteristic.
A kind of crystal methamphetamine (methamphetamine) detection method based on SPR technique is protected in patent 201610878373.8,
The inside is indirect competitive, the antigen secure bond of crystal methamphetamine on SPR chip, while flowing into crystal methamphetamine
Monoclonal antibody and sample to be tested, make crystal methamphetamine antigen and antibody combine, competed the combination of small molecule and antibody,
To detect the content height of small molecule crystal methamphetamine.The instrument that this method uses is that (instrument is more by Biacore 3000
It is suitable for the combination of macromolecular and macromolecular), CM5 chip is used, although indirect competitive high sensitivity, because fixed
Be methyl isopropylamine antigen, process is more cumbersome than direct competition method, and every time detection need to consume antibody, higher cost.
In view of agricultural product routine testing sample size is larger and the fresh and alive characteristic of the edible agricultural product such as fruits and vegetables, conventional agriculture are residual
Instrument analytical method can not meet the demand that a large amount of samples complete rapid screening within a short period of time.Therefore, people urgently wish
Prestige has a kind of easy, quick, residual detection technique of agriculture that is inexpensive and containing much information to can be carried out large batch of Screening tests.
Summary of the invention
The technical problem to be solved in the present invention is to provide the bases of thiacloprid residual quantity in a kind of fast slowdown monitoring agricultural and sideline product of energy
In the insecticide thiacloprid method for detecting residue of SPR technique.
In order to solve the above technical problem, the present invention provides a kind of, and the nicotinic insecticide thiacloprid based on SPR technique is residual
Stay detection method, successively the following steps are included:
Step (1): SPR chip (CM7 chip) (is placed into SPR instrument as sensing chip) as sensing chip；
Step (2): correction circulation passage washs SPR chip with buffer (PBS-P+ buffer)；
Step (3): being passed through the EDC/NHS mixed liquor of 200~400 μ L toward SPR chip surface, and flow velocity is 10~30 μ L/
Min, to activate the carboxylic group of SPR chip surface；
In the EDC/NHS mixed liquor, EDC (1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride) concentration
It is (0.1 ± 0.01) M for (0.4 ± 0.04) M, NHS (n-hydroxysuccinimide) concentration；It takes water as a solvent；
The EDC/NHS mixed liquor the preparation method comprises the following steps: first distinguish compound concentration be (0.8 ± 0.08) M EDC aqueous solution,
Concentration is the NHS aqueous solution of (0.2 ± 0.02) M, then mixes above-mentioned EDC aqueous solution, NHS aqueous solution according to the volume ratio of 1:1
It closes, obtains EDC/NHS mixed liquor；
Step (4): 0.5~2.0 μ L/min thiophene is flowed on the SPR chip after the resulting activating surface carboxyl of step (3)
Worm quinoline monoclonal antibody (SCL-Ab) dilution, is coupled to it on carboxyl activated, until SPR chip is saturated；
Thiacloprid monoclonal antibody dilution are as follows: use the sodium acetate buffer solution of pH4.0~5.5 (the 10mM second of preferably pH5.0
Sour sodium buffer solution) dilution thiacloprid monoclonal antibody to concentration be 20 ± 5 μ g/ml, obtain thiacloprid monoclonal antibody dilution；
Step (5): with the stream of 10~30 μ L/min on the SPR chip after the resulting thiacloprid antibody coupling of step (4)
Speed is passed through 70~100 μ L ethanolamine hydrochloric salt solution (concentration 1-2M), to close the unreacted carboxyl site activated；
Step (6): PBS-P is used+Thiacloprid raw medicine is diluted to the thiacloprid solution of series of concentrations, 10~30 μ by buffer
L.min-1Sample introduction (120 ± 20) s (thus combination of realization and thiacloprid antibody), then by (10 ± 2) mM NaOH with 10~30 μ
L.min-1Flow velocity flow through chip surface so that regeneration after RU value with combine thiacloprid solution before RU value it is identical (regenerate about
300s, that is, the time that (10 ± 2) mM NaOH flows through chip is about 300s), then according to RU changing value before and after association reaction with
Thiacloprid concentration establishes standard curve；
Step (7): 30~50 μ L samples to be tested are taken, with 10~30 μ L.min-1Flow velocity be passed through SPR chip, then will (10 ±
2) mM NaOH is with 10~30 μ L.min-1Flow velocity flow through chip surface so that regeneration after RU value and combine thiacloprid solution before
RU value it is identical (regeneration about 300s, that is, the time that (10 ± 2) mM NaOH flows through chip is about 300s), and according to association reaction
Front and back RU changing value establishes standard curve with thiacloprid concentration and (that is, recording RU changing value before and after association reaction, brings step into
(6) resulting standard curve), obtain the content of thiacloprid in sample to be tested.
Improvement as the nicotinic insecticide thiacloprid method for detecting residue of the invention based on SPR technique:
In the step (6), the thiacloprid solution of series of concentrations is 0.78125,1.56,3.125,6.25,12.5nM.
Further improvement as the nicotinic insecticide thiacloprid method for detecting residue of the invention based on SPR technique:
The resulting calibration curve equation of the step (6) is y=33.33-32.28/ (1+ (x/8.42)2.36)。
As the further improvement of the nicotinic insecticide thiacloprid method for detecting residue of the invention based on SPR technique,
In the step (2):
Circulation passage is corrected with the glycerine water solution that volumetric concentration is (70 ± 5) %；
Buffer is PBS-P+Buffer, the PBS-P+Buffer is delayed by polysorbate surfactant P20 and phosphate
Solution (PBS, 0.01M, pH 7.4) is rushed to be obtained by mixing；The PBS-P+The body of polysorbate surfactant P20 in buffer
Product concentration is 0.05% (v/v)；
PBS-P+Buffer washs chip surface with the flow velocity of (10 ± 1) μ L/min, and wash time is (200 ± 20) s.
Further improvement as the nicotinic insecticide thiacloprid method for detecting residue of the invention based on SPR technique:
The thiacloprid antibody is the source of mouse monoclonal antibody with specific recognition prepared by immunogene of SCL-BSA.
Further improvement as the nicotinic insecticide thiacloprid method for detecting residue of the invention based on SPR technique:
SCL-BSA immunogene is prepared by thiacloprid haptens (SCL-hpten) coupling bovine serum albumin BSA.
As the further improvement of the nicotinic insecticide thiacloprid method for detecting residue of the invention based on SPR technique,
SCL haptens the preparation method comprises the following steps:
Thiacloprid, cesium carbonate, mercaptopropionic acid are dissolved in DMSO with the molar ratio of 1:0.2~2.0:0.2~2.0,20
It is stirred to react at~100 DEG C 8~10 hours, reaction solution pH is adjusted to (2.0 ± 0.2), and ethyl acetate extraction (can extract for ethyl acetate
Merge organic phase after taking 3 times), organic phase passes through anhydrous Na2SO4Crude product is concentrated under reduced pressure to give after drying, by silica gel column purification
(EA:PE:HCOOH=80:20:1) SCL haptens (white solid product) is obtained after.
As the further improvement of the nicotinic insecticide thiacloprid method for detecting residue of the invention based on SPR technique,
Sample to be tested in step (7) can be plant sample, pedotheque, water sample.
The present invention, using antibody as sensor sensing recognition component, is utilized using antigen-antibody immune response as basic principle
Direct non-competing method (quickly, conveniently) SPR technique carries out residue detection to insecticide thiacloprid, by chemical bond by thiacloprid list
Clonal antibody is fixed on SPR chip surface, causes signal intensity in conjunction with the thiacloprid small molecule in prepare liquid.
The present invention starts with from the synthesis of haptens, synthesizes the good haptens of purity is high, immunity, is made into BSA coupling
Immunogene obtains the high monoclonal antibody of specificity after small white mouse is immunized, is then directly coupled with SPR chip with this antibody, can
Directly to detect the content of testing sample solution small molecular thiacloprid.
In the synthesis process of haptens of the present invention, acid binding agent is done with cesium carbonate substitution potassium hydroxide, yield is higher.
In order to overcome existing analysis method there are complicated for operation, matrix interference is more, analysis data deviation the problems such as, the present invention
A kind of quick monitoring method based on SPR technique is established, with the residual of nicotine pesticide in quick, accurate detection vegetables.
The demand that the present invention is monitored using Agricultural Products quality safety is point of penetration, to apply more and more extensive anabasine
Class pesticide thiacloprid is detection target, researchs and develops a set of Fast Determination of Pesticide Residue technology based on SPR, outstanding for agricultural product
It is tealeaves, the remaining quick detection of pesticide in water fruits and vegetables and other Agricultural Samples, is quickly detected for correlative study and pesticide
Technical support is provided.The residual rapid screening of agriculture for really realizing batch samples, to improve the agricultural production quality of our province, China
It is horizontal to measure security control, ensures that people's health has extremely important realistic meaning.
At present both at home and abroad there is not yet thiacloprid residue detection and the combined report of SPR technique.
The invention has the following beneficial effects:
(1), the method for the invention is easy to operate, in real time, quickly, it is only necessary to sample to be tested is passed through SPR instrument,
Real-time observed result.
(2), chip preparation method of the present invention is mature, has been commercialized, and purchase is convenient, fixes the immune of antibody
Chip may be reused up to a hundred times, be a kind of very economical detection method.
(3), the method for the present invention sensitivity achieved is higher, and detection time is shorter, is that the general prior art cannot all compare
Quasi-.
(4), since the sensitivity of common liquid chromatography, Liquid Chromatography/Mass Spectrometry detection thiacloprid only has 10-100ng/mL, when
When measuring samples content is lower than its sensitivity, general liquid chromatogram and LC-MS instrument will be unable to detect, the immune glue of thiacloprid
Body gold test paper strip also in exploitation, is not sufficiently stable, and the present invention just solves the problems, such as this.
The present invention applies SPR chip technology for the first time, by fixing thiacloprid monoclonal antibody on chip, in Direct Recognition point
Analyse the residual quantity of thiacloprid raw medicine in test sample.Detection sensitivity is high, minimum detectability 0.197ng/mL, EC50For
2.13ng/mL, detection range are detected sensitiveer between 1.18-2.81ng/mL using SPR.It is another: to be used in existing literature at present
Immunization ELISA, lowest detection are limited to 0.47 μ g/L (g/mL) n, EC50For 10ng/mL, (0.01mg/L).
That is, the present invention directly highly sensitive can not detect thiacloprid pesticide molecule and agricultural and sideline product sample for existing
The problem of product complex pretreatment, thiacloprid can directly be detected using SPR sensorgram technology using direct non-competing method by providing one kind
Method.The step is simple, highly sensitive, high specific, can in fast slowdown monitoring agricultural and sideline product thiacloprid residual quantity method.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 is SPR dynamics sensing figure (grey level) and matched curve (black line).
Thiacloprid concentration from top to bottom is respectively 12.5nM, 6.25nM, 3.125nM, 1.56nM, 0.78125nM (effective
Concentration)；
Light line is matched curve, and dark line is measured curve.
Fig. 2 is standard curve of the thiacloprid antibody in conjunction with thiacloprid.
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
The main agents information mentioned in following embodiment is shown in Table 1, and key instrument and facility information are shown in Table 2.
Table 1, main agents information
Table 2, key instrument and equipment
SPR chip used by following embodiment is the CM7 chip of GE company production, which is carboxy methylation Portugal
Glycan, used SPR instrument are the Biacore T200 of GE formula production.
Basic preparation before embodiment 1, SPR
1. pesticide molecule hapten molecule, which designs and its synthesize-directly decides to be immunized, obtains the recognition capability of antibody:
The fundamental factor for influencing immunochemistry analysis quality is the selectivity (or specificity) and compatibility of antibody, these property
Matter is decided by the structure of immune hapten molecule again, and therefore, the MOLECULE DESIGN of immune haptens is to establish small molecule to exempt from synthesis
The committed step of epidemic disease chemical analysis method.So hapten molecule design and synthesis are the difficult points in emphasis in the present invention.According to
The molecular structure and molecule feature of existing pesticide, design and select suitable route, have synthesized thiacloprid haptens, structure warp
It crosses1H NMR and MS are accredited as correctly.The present invention using made of cesium carbonate acid binding agent be it is new, yield is higher.
The synthesis of 1.1 thiacloprid haptens
1) reaction equation
2) reaction process
Sequentially added into 50mL reaction flask thiacloprid (2.53g, 10mmol), DMSO (15mL), cesium carbonate (4.89g,
15mmol) and then reaction is placed at 80 DEG C and is stirred to react 8~10 by mercaptopropionic acid (1.272g, 12mmol), displacement nitrogen
Hour (preferably 9 hours).To after reaction, reaction solution pH is adjusted to 2 or so (hydrochloric acid solutions for using concentration as 10-15%
It is adjusted), (dosage of each ethyl acetate is 50ml) merges organic phase after ethyl acetate extracts 3 times, and organic phase passes through nothing
Water Na2SO4It is concentrated under reduced pressure after drying and (uses water vacuum pump pressure, be concentrated to dryness under pressure about 50MPa) and obtain crude product.
Crude product is passed through into silica gel column purification: selecting the Conventional glass chromatographic column of 2.5cm (internal diameter) * 50cm (height), it is built-in
The silica gel 40g of 200-300 mesh；Silica gel after the crude product acetone solution of 2ml with 100-200 mesh is mixed uniformly, is dried, loading,
It is flowed using EA (ethyl acetate): the mixed liquor of PE (petroleum ether): HCOOH (formic acid)=80:20:1 volume ratio as eluant, eluent, control
Speed is 10mL/min-50mL/min；TLC tracking, collection meet the eluent that TLC shows only one compound；By eluent
Merge concentration post-processing；Obtain 2.34g (yield: 73%) white solid product, as thiacloprid haptens (SCL-hp),
Than literature procedure (replacing cesium carbonate with potassium hydroxide), yield improves 40% and (when using potassium hydroxide, receives
Rate is only 33% or so).
3) structural confirmation
Nuclear magnetic resonance spectroscopy, carbon spectrum and three kinds of methods of high resolution mass spectrum determine that structure is correct:1H NMR(500MHz,DMSO)
δ 8.40 (s, 1H, N=CH), 7.58 (dd, J=8.3,2.3Hz, 1H, CH), 7.31 (d, J=8.3Hz, 1H, CH), 4.57 (s,
2H,CH2), N 3.89 (t, J=7.6Hz, 2H, CH2), N 3.48 (t, J=7.6Hz, 2H, SCH2), 3.30 (t, J=7.0Hz, 2H,
SCH2), 2.64 (t, J=7.0Hz, 2H, CH2COO)；13C NMR(126MHz,DMSO)δ175.00,173.39,158.03,
149.58,137.12,127.31,122.35,117.76,53.17,47.06,34.48,27.77,25.05；High resolution mass spectrum
HRMS:321.5555 (322.06).
The preparation (conventionally) of 1.2 thiacloprid immunizing antigens:
Amino covalence coupling on carboxyl and BSA on haptens SCl-hp is prepared by immunogene using active ester method；Side
Method summary are as follows:
0.3mmol NHS (being dissolved in 0.1mL DMF) is added in 0.1mmol haptens (being dissolved in 0.8mL DMF), is stirred to react
15min is mixed；0.15mmol DCC (being dissolved in 0.1mL DMF) is added dropwise again, is stirred overnight at room temperature, is protected from light.Reaction solution
After 4000r/min is centrifuged 10min, the supernatant of 300 μ L is drawn, the coupling for being slowly dropped to 3mL dissolved with 60mg BSA buffers
In liquid CBS (0.01mol/L), reaction 4h is stirred at room temperature；To after the reaction was completed, be packed into bag filter (molecular cut off 60-
66KDa), 0.01mol/L, the PBS of pH 7.4 are then first used with distilled water 2 times (each dialysis time is about 120 minutes) of dialysis
Dialyse 3d, takes out packing, saves in -20 DEG C.
Amino covalence coupling on carboxyl and OVA on haptens SCl-hp is prepared by coating antigen using mixed anhydride method；
Method summary are as follows: 0.125mmol haptens is dissolved in 1mL DMF, and the positive tri-n-butylamine of 30 μ L and 15 μ L chloro-carbonic acids are added under stirring condition
Reaction 1h is stirred at room temperature in ethyl ester.It draws 850 μ L reaction solutions and coupling buffer CBS to 8mL dissolved with 160mg OVA is slowly added dropwise
In (0.01mol/L), reaction 3h is stirred at room temperature；Through dialysis treatment, (molecular cut off is 40-45k Da to reaction solution, uses distilled water
Dialysis, dialysis time is about 120 minutes) after, -20 DEG C of preservations.
1.3 animal immune schemes (conventionally):
50 μ g immunogene normal saline dilutions to 50 μ L, with the quick adjuvant of isometric QuickAntibody before injection
It quickly mixes well, injects Balb/c mouse Calf muscle position, every 100 μ L, if three reprocessings.3rd week, the 5th week
Same dose booster immunization.7d after second of booster immunization takes mouse tail blood to survey antiserum titre (with sero-fast extension rate
Meter).The measurement of potency uses indirect enzyme-linked immunosorbent assay (Indirect enzyme linked immunosorbent
Assay, i-ELISA method), envelope antigen (coating antigen) concentration is 10 μ g/mL, with OD490nmIt is sero-fast dilute when being worth close to 1.0
Releasing multiple is its potency, selects the mouse that potency is high, strong to small molecule compound recognition capability to carry out end and exempts from (final immunization), makees
Preparation for immune spleen cell source, for hybridoma.
The preparation (conventionally) of 1.4 hybridomas and monoclonal antibody:
Cell fusion and selection culture:
After 3d is exempted from mouse end, the preparation of immune spleen cell is carried out.Immune spleen cell and murine myeloma cell (SP2/0)
Integration percentage is advisable (preferably 6:1) with 5~10:1, and wherein myeloma cell tames through 8-AG in advance, it is ensured that quick to methotrexate (MTX)
Sense, and viable count is higher than 95%.Entire fusion process need to be carried out aseptically, and the cell of fusion is further selected through HAT
Select culture medium culture.
Cell screening:
12~14d after fusion, survival and the hybridoma bred further are screened through indirect elisa method.Antigen
Peridium concentration is 10 μ g/mL, and screening can specific recognition artificial antigen (OD490nm>=0.5), OVA is identified without intersection, while right
The identification high sensitivity of 1 μ g/mL analyte carries out the expansion culture of 24 holes in 50% hybridoma cell strain；To long to hole area
When one third, further screening obtains the strong hybridoma of high sensitivity, secretion capacity；Through 4~6 wheel limiting dilutions, most
The hybridoma cell strain that can secrete high affinity monoclonal antibody is obtained eventually.It periodically freezes and recovery cell strain, inspection antibody divides
Situation is secreted, antibody affinity is lost caused by preventing because of cytometaplasia.
Monoclonal antibody preparation:
The hybridoma for the stably excreting monoclonal antibody that screening is obtained expands culture to logarithmic phase.The injection of F1 generation mouse peritoneal
0.5mL norphytane, one to inoculating cell after two weeks, 0.4mL about 1.0 of every mouse peritoneal injection after brine ×
106A cell.7~10d extracts ascites after inoculation, obtains monoclonal antibody, dialysis by octanoic acid-ammonium sulfate precipitation method purifying
- 20 DEG C of preservations afterwards.
1.5 indirect enzyme-linked immunosorbent assays are established:
Response effect after evaluating mouse immune using indirect elisa method and indirect competitive ELISA method (ic-ELISA), with
And hybridoma cell strain high for screening sensitivity, that secretion capacity is strong.Method is as follows: artificial antigen is dilute with coating buffer
It releases to 10 μ g/mL, using carrier protein OVA as antigen-negative controls, 4 DEG C are coated with overnight (100 hole μ L/).Next day reacts on 37 DEG C
It carries out, the PBS (every 300 hole μ L/ of hole) that 2.0% skim milk is added closes 30min；The diluted antiserum of PBS or cell is added
Supernatant (100 hole μ L/) reacts 1h；Diluted rabbit anti-mouse igg-the HRP of PBS (1:40,000,100 hole μ L/) is added and reacts 45min；
Every step reaction is completed to be both needed to washing buffer board-washing 3 times.Finally, the substrate reactions liquid (100 hole μ L/) of Fresh is added,
37 DEG C of incubation 15min are added and terminate reaction solution (50 hole μ L/), measure each hole with SpectraMax i3 multi-function microplate reader
OD490nmValue.
Under same antigen peridium concentration, while (2 times of work are dense for 50 μ L/ hole test analytes of addition and 50 hole μ L/ antibody
Degree), analyte and envelope antigen competitive binding antiserum or cell conditioned medium, other reaction conditions are constant, further evaluate it
To the recognition capability of small molecule compound.
Thiacloprid raw medicine and thiacloprid antibody binding capacity:
Thiacloprid is monoclonal antibody-purified and be dissolved in 10mM phosphate buffered saline solution (PBS) to ultimate density be 1mg/
mL.Thiacloprid antibody activity is measured by noncompetitive indirect ELISA (iELISA), and is measured by competitiveness iELISA to thiophene
The sensibility (50,50% inhibition concentration of IC) of worm quinoline: thiacloprid haptens-OVA is used as coating detection antigen.EC50=
13.4ppb shows that antibody has the compatibility of height to thiacloprid.Since ELISA only provides the immune response under equilibrium condition
Final data need to obtain from SPR about dynamic information.
2 SPR of embodiment analysis
1) SPR instrument, is opened, SPR chip (CM-7) is placed into Biacore T200 system, chip pocket is locked；
SPR instrument temperature is set as 25 DEG C；70% (w/w) glycerite corrects circulation passage.Buffer is PBS-P+ buffer, that is, is contained
There is the phosphate buffer solution (PBS, 0.01M, pH 7.4) of 0.05% (v/v) polysorbate surfactant P20, with 10 μ L/
The flow velocity of min washs chip surface 200s.
The PBS-P+Buffer is by polysorbate surfactant P20 and phosphate buffer solution (PBS, 0.01M, pH
7.4) it is obtained by mixing；The PBS-P+The volumetric concentration of polysorbate surfactant P20 is 0.05% (v/v) in buffer.
2), anti-using the 10mM sodium acetate buffer solution dilution thiacloprid monoclonal of pH (4.0,4.5,5.0 and 5.5) respectively
Body, until concentration is 20 μ g/ml, sample introduction is primary respectively, and flow is 200-300 μ L；When pH is 5.0, thiacloprid antibody is in chip
Surface preenrichment concentration highest (Ru value is maximum), thus select the 10mM sodium acetate buffer of pH5.0 solid as thiacloprid antibody
Fixed dilution buffer.
3) EDC/NHS of (injection) 400 μ L, is passed through toward the resulting SPR chip surface of step 1) with the flow velocity of 20 μ L/min
Mixed liquor, to activate the carboxylic group of SPR chip surface；
In the EDC/NHS mixed liquor, EDC (1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride) concentration
For 0.4M, NHS (n-hydroxysuccinimide) concentration is 0.1M, is taken water as a solvent；
The EDC/NHS mixed liquor the preparation method comprises the following steps: EDC aqueous solution that first compound concentration is 0.8M respectively, concentration are
Then above-mentioned EDC aqueous solution, NHS aqueous solution are mixed according to the volume ratio of 1:1, it is mixed to obtain EDC/NHS by the NHS aqueous solution of 0.2M
Close liquid；
4) thiacloprid, is passed through with the flow velocity of 20 μ L/min on the SPR chip after the resulting activating surface carboxyl of step 3)
Monoclonal antibody is coupled to it on carboxyl activated；The thiacloprid of channel 2 and 10mM sodium acetate buffer (pH 5.0) are anti-
Body coupling, rest channels inactivation, channel 1 is used as blank control (thiacloprid antibody-solutions are not added).Reach saturation after about 900s.
Specifically: the resulting thiacloprid monoclonal antibody of embodiment 1 is diluted extremely with the 10mM sodium acetate buffer solution of pH5.0
Concentration is 20 μ g/ml, obtains thiacloprid monoclonal antibody dilution, is passed through this dilution in channel 2；With activate on SPR chip
Carboxyl coupling, participates in reaction.
5), it is passed through 100 μ L's on the SPR chip of the resulting coupling thiacloprid antibody of step 4) with the flow velocity of 20 μ L/min
The ethanolamine hydrochloric salt solution (pH8.5) of 1M handles 300s, to close the unreacted carboxyl site activated；
Use buffer PBS-P+After rinsing 300s, it is ready for SPR analysis.
Embodiment 3, antibody dynamics/affinity detection
Use PBS-P+Buffer thiacloprid raw medicine is diluted to a series of various concentrations thiacloprid solution (0.195nM,
0.391nM, 0.78125nM, 1.56,3.125,6.25,12.5nM, 25.0nM, 50nM)；
The thiacloprid solution of every kind of concentration is performed the following operation respectively:
30μL.min-1Sample introduction is to the resulting SPR chip of embodiment 2, sample injection time 120s, to realize and thiacloprid
The combination of monoclonal antibody, by 10mM NaOH with 30 μ L.min-1Flow velocity flow through chip surface, regenerate 300s (that is, 10mM
NaOH flows through the time of chip for 300s), (can be with original one so that the RU value after regeneration is identical as before combination thiacloprid solution
It causes).
The response in 2 channels is subtracted to the response of reference channel 1, excludes the non-specific knot in buffer or sample
It closes, excludes bulk refractive index change caused by buffer or sample.Blank control (zero-dose) is also contained thiacloprid from each
Sample in deduct.That is, the response that the response in channel 2 subtracts reference channel 1 is the actual signal of each sample.Zero-dose
Deduction is exactly the actual signal of each thiacloprid concentration samples, deducts the practical letter for the blank control sample that thiacloprid concentration is zero
Number.
Acquired results are as shown in Figure 1.Note: effective concentration within the scope of instrument is 0.78125nM, 1.56nM, 3.125nM,
6.25nM,12.5nM。
In Fig. 1, it is sample injection time that sample enters instrument that abscissa is corresponding；The RU value of ordinate is that thiacloprid antibody exists
Corresponding response on SPR chip.Grey curves are empirical curve, that is, the thiacloprid solution of various concentration and the combination of antibody
Curve；Black curve is matched curve, i.e., instrument Rigen is according to binding pattern, the numerical value or standard curve of the fitting of response size.
kaThe rate combined between analyte and ligand, kdFor the rate of dissociation, affinity KDBy kinetic constant ka,kd
It obtains, KD=kd/ka is the synthesis result of two processes of association and dissociation.In general, KDValue is 10-7To 10-10When M, table
Reveal good affinity (being specifically shown in document 1), and K in this experimentDValue is 1.545 × 10-9, thus show good affine
Power.And combination stability and kdValue is related, kdSmaller combination is more stable.K in this experimentdValue is 5.308 × 10-4, with Guo
4.30×10-4Numerical value is close, thus combines more stable.
The binding kinetics of table 3, thiacloprid antibody and thiacloprid characterize
Note: document 1, Yakes, B.J.；Kanyuck,K.M.；DeGrasse,S.L.,First Report of a
Direct Surface Plasmon Resonance Immunosensor for a Small Molecule Seafood
Toxin.Analytical Chemistry 2014,86,(18),9251-9255。
Embodiment 4, sensitivity for analysis, the foundation of calibration curve:
Use PBS-P+Buffer thiacloprid raw medicine is diluted to a series of various concentrations thiacloprid solution (0.195,
0.391,0.78125,1.56,3.125,6.25,12.5,25.0,50nM), 30 μ L.min-1Sample introduction 120s, sample introduction 9 samples, 1
A zero-dose blank.
Mode of operation is equal to embodiment 3.
Calibration curve gradient concentration range is 0.3906-100nM, and concentration is 0 as blank control.Sample solution is with 30 μ
The flow velocity sample introduction 120s of L.min-1, then by 10mM NaOH with 30 μ L.min-1Flow velocity flow through chip surface 300s (to realize
Dissociation and regeneration).The response in 2 channels is subtracted into 1 channel of reference channel, excludes the non-specific binding in buffer or sample
From bulk refractive index change caused by buffer or sample.Standard sigmoidal curve (Fig. 2) is made using OriginPro 8.5.
To obtain y=33.33-32.28/ (1+ (x/8.42)2.36)；Wherein, EC50=8.42nM (about 2.1278ppb),
The range of linearity: EC20-EC80=4.679-11.12nM, thus at 4.679-1.112nM (1.18-2.81ng/mL), it uses
SPR detection is sensitiveer.
Embodiment 5, actual sample detection
1, water sample is handled:
Rice field water is taken, filtered rice field water sample 100mL is accurately measured, is placed in 250mL separatory funnel, uses acetic acid respectively
Ethyl ester 30mL is extracted twice, and discards water phase, merges organic phase, and 50 DEG C of waters bath with thermostatic control, which are concentrated into, closely to be done, respectively at different dissolutions
Sample introduction after reason:
1) it is dissolved with the PBS-P+ of 5 or 10mL, and (the 0.22- μm of film) sample liquids of filtering is subjected to SPR analysis；
2) it is settled to 2.0mL with acetonitrile-water (3: 7), crosses 0.45 μm of filter membrane, measured into HPLC.
2, vegetable sample is handled:
Taking vegetables/fruit sample that QuEChERS method is respectively adopted respectively, (this is widely used pesticide residue analysis sample
Product pre-treating method):
For effective component extracting, 10mL acetonitrile is added separately in tomato (10g) sample, vortex mixed is then used
Device is aggressively shaken 2 minutes.Then, NaCl (1.0g) and anhydrous MgSO4 (4.0g) is added in mixture.Shake 1 minute and from
After ten minutes, whole upper layers (about 4.0mL) is transferred in 10mL centrifuge tube for the heart, and 100mg brothers amine is contained in centrifuge tube
(PSA) whole supernatants (about 0.5mL) is transferred to after continuing shake 30 seconds and being centrifuged 1 minute with the anhydrous MgSO4 of 0.6g
In 10mL glass tube and in N2It is evaporated at 40 DEG C under air-flow, as needed by dry residue:
1) it is redissolved with the PBS-P+ of 5 or 10mL, and (the 0.22- μm of film) sample liquids of filtering is subjected to SPR analysis
2) dried object is settled to 2.0mL with acetonitrile-water (3: 7), crosses 0.45 μm of filter membrane, measures into HPLC.
Above-mentioned resulting every kind of sample liquids are operated as follows excessively respectively:
30μL.min-1Sample introduction is to the resulting SPR chip of embodiment 2, sample injection time 120s, to realize and thiacloprid
The combination of monoclonal antibody, by 10mM NaOH with 30 μ L.min-1Flow velocity flow through chip surface, regenerate 300s (that is, 10mM
NaOH flows through the time of chip for 300s), (can be with original one so that the RU value after regeneration is identical as before combination thiacloprid solution
It causes).
It is detected according to method described in above-described embodiment 3, specifically: by resulting " sample liquids " alternate embodiment
3 " the thiacloprid solution of serial various concentration ", remaining is equal to embodiment 3；Ru value (Y value) is directly obtained from instrument,
Then the above results are substituted into y=33.33-32.28/ (1+ (x/8.42) respectively2.36), to obtain sample accordingly
The thiacloprid residual quantity X (unit nM/L) of product liquid.According to thiacloprid molecular weight, thiacloprid residual concentration μ g/kg is obtained.
Measure rice field water sample with liquid chromatography technology and the vegetables tomato sample bought in the market in thiacloprid content be
It is close with SPR content detection result.Specifically it is shown in Table 4
Thiacloprid residual quantity SPR and HPLC testing result compare in 4 agricultural and sideline product of table
ND(not detectable),less than the corresponding LOD
Note: μ g/kg, i.e. ppb.
In short, quickly, sensitive diagnostic method, this is that first passage is noncompetitive straight the invention proposes a kind of simple
The method for connecing SPR immune sensing detection trace thiacloprid.The test has specificity and sensibility, to thiacloprid and sensor core
The LOD of piece is 0.197ng mL-1.The application of actual life test sample shows that this new immune sensor makes in which can be convenient
With the reliable tools agricultural product of thiacloprid residue in monitoring environment.This research is also laid a good foundation for development for other
The noncompetitive SPR immunosensor of LMW compound.
The above list is only a few specific embodiments of the present invention for finally, it should also be noted that.Obviously, this hair
Bright to be not limited to above embodiments, acceptable there are many deformations.Those skilled in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (8)
1. the nicotinic insecticide thiacloprid method for detecting residue based on SPR technique, it is characterised in that the following steps are included:
Step (1): using SPR chip as sensing chip；
Step (2): correction circulation passage washs SPR chip with buffer；
Step (3): being passed through the EDC/NHS mixed liquor of 200~400 μ L toward SPR chip surface, and flow velocity is 10~30 μ L/min, from
And activate the carboxylic group of SPR chip surface；
In the EDC/NHS mixed liquor, EDC concentration is that (0.4 ± 0.04) M, NHS concentration is (0.1 ± 0.01) M；It is molten with water
Agent；
Step (4): 0.5~2.0 μ L/min thiacloprid is flowed on the SPR chip after the resulting activating surface carboxyl of step (3)
Monoclonal antibody dilution, is coupled to it on carboxyl activated, until SPR chip is saturated；
Thiacloprid monoclonal antibody dilution are as follows: dilute thiacloprid monoclonal antibody with the sodium acetate buffer solution of pH4.0~5.5
It is 20 ± 5 μ g/ml to concentration, obtains thiacloprid monoclonal antibody dilution；
Step (5): logical with the flow velocity of 10~30 μ L/min on the SPR chip after the resulting thiacloprid antibody coupling of step (4)
Enter 70~100 μ L ethanolamine hydrochloric salt solution, to close the unreacted carboxyl site activated；
Step (6): PBS-P is used+Thiacloprid raw medicine is diluted to the thiacloprid solution of series of concentrations, 10~30 μ L.min by buffer-1Sample introduction (120 ± 20) s, then by (10 ± 2) mM NaOH with 10~30 μ L.min-1Flow velocity flow through chip surface so that regeneration
RU value afterwards is identical as the RU value before thiacloprid solution is combined, then according to RU changing value before and after association reaction and thiacloprid concentration
To establish standard curve；
Step (7): 30-50 μ L sample to be tested is taken, with 10~30 μ L.min-1Flow velocity be passed through SPR chip, then by (10 ± 2) mM
NaOH is with 10~30 μ L.min-1Flow velocity flow through chip surface so that regeneration after RU value and combine thiacloprid solution before RU
It is worth identical, and establishes standard curve according to RU changing value before and after association reaction and thiacloprid concentration, obtain thiophene in sample to be tested
The content of worm quinoline.
2. the nicotinic insecticide thiacloprid method for detecting residue according to claim 1 based on SPR technique, feature exist
In:
In the step (6), the thiacloprid solution of series of concentrations is 0.78125,1.56,3.125,6.25,12.5nM.
3. the nicotinic insecticide thiacloprid method for detecting residue according to claim 1 or 2 based on SPR technique, special
Sign is: the resulting calibration curve equation of the step (6) is y=33.33-32.28/ (1+ (x/8.42)2.36)。
4. any nicotinic insecticide thiacloprid method for detecting residue based on SPR technique according to claim 1~3,
It is characterized in that in the step (2):
Circulation passage is corrected with the glycerine water solution that volumetric concentration is (70 ± 5) %；
Buffer is PBS-P+Buffer, the PBS-P+Buffer is molten by polysorbate surfactant P20 and phosphate-buffered
Liquid is obtained by mixing；The PBS-P+The volumetric concentration of polysorbate surfactant P20 is 0.05% in buffer；
PBS-P+Buffer washs chip surface with the flow velocity of (10 ± 1) μ L/min, and wash time is (200 ± 20) s.
5. the nicotinic insecticide thiacloprid method for detecting residue according to any one of claims 1 to 4 based on SPR technique,
It is characterized by:
The thiacloprid antibody is the source of mouse monoclonal antibody with specific recognition prepared by immunogene of SCL-BSA.
6. the nicotinic insecticide thiacloprid method for detecting residue according to claim 5 based on SPR technique, feature exist
In:
SCL-BSA immunogene is prepared by thiacloprid haptens SCL-hpten coupling bovine serum albumin BSA.
7. the nicotinic insecticide thiacloprid method for detecting residue according to claim 5 or 6 based on SPR technique, special
Sign be SCL haptens the preparation method comprises the following steps:
Thiacloprid, cesium carbonate, mercaptopropionic acid are dissolved in DMSO with the molar ratio of 1:0.2~2.0:0.2~2.0,20~100
It is stirred to react at DEG C 8~10 hours, reaction solution pH is adjusted to (2.0 ± 0.2), and ethyl acetate extraction, organic phase passes through anhydrous Na2SO4
It is concentrated under reduced pressure to give crude product after drying, obtains SCL haptens after silica gel column purification (EA:PE:HCOOH=80:20:1).
8. any nicotinic insecticide thiacloprid method for detecting residue based on SPR technique according to claim 1~7,
It is characterized by: the sample to be tested in step (7) is plant sample, pedotheque, water sample.
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