CN105061343A - Tri(2,3-dibromopropyl)triallyisocyanurate hapten, and preparation and application - Google Patents
Tri(2,3-dibromopropyl)triallyisocyanurate hapten, and preparation and application Download PDFInfo
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Abstract
The invention discloses a tri(2,3-dibromopropyl)triallyisocyanurate hapten, and preparation and application. The tri(2,3-dibromopropyl)triallyisocyanurate hapten has a structural formula described in the specification. A preparation method for the tri(2,3-dibromopropyl)triallyisocyanurate hapten comprises the following steps: using 2,4,6-(allyloxyl)-1,3,5-triazine as a raw material, and carrying out two-step reaction so as to prepare a TBC hapten with a secondary amino active group. The tri(2,3-dibromopropyl)triallyisocyanurate hapten has the advantages of simple and convenient preparation method, good stability, low cost and easy industrialized production; and the tri(2,3-dibromopropyl)triallyisocyanurate hapten is prepared into a tri(2,3-dibromopropyl)triallyisocyanurate artificial antigen by coupling with a carrier protein through a glutaraldehyde method or a carbodiimide method or a diazotization method, and provides guarantees for preparing an antibody with good specificity and high titer and establishing an avidin-biotinylated enzyme-linked immunosorbent assay method.
Description
Technical field
The present invention relates to haptens and preparation method thereof, be specifically related to a kind of three (2,3-dibromopropyl) isocyanuric acid ester haptens and preparation thereof, purposes.
Background technology
Bromide fire retardant is one of organic fire-retardant that at present output is maximum in the world, wherein mainly Poly Brominated Diphenyl Ethers (PBDEs) and Polybrominated biphenyl (PBBs) class material, in addition wherein triazines in addition.Wherein, three (2,3-dibromopropyl) isocyanuric acid ester (Tris (2,3-dibromopropyl) isocyanurate, TBC), containing six bromine atoms in molecular structure, be the main products of the current organic bromine flame retardant of China, belong to the agent of triazine fire-retardant.As the new variety of fire retardant, TBC has excellent flame retardant properties, thermostability, the not easily advantage such as photodissociation, low smokiness, be widely used in the goods neutralization materials fields such as fibre reinforced plastics, agricultural polyurethane film, polyolefine, polyvinyl chloride (PVC), polyphenyl alkene, ABS resin unsaturated polyester, synthetic rubber and synthon, and along with the use of these products, slowly spill in environment.
Domestic and international vast environment scholar has done large quantity research to the concentration of TBC in various surrounding medium and biological sample and toxic effect.Research shows, TBC can exist in the various surrounding mediums such as various water body, bed mud, soil, air, can discharge toxic gas hydrogen bromide when cracking occurs TBC, and the moisture that hydrogen bromide easily absorbs in air forms Hydrogen bromide, there is very strong corrodibility, there is secondary harm.Its octanol-water partition coefficient (LogKow, 25 DEG C) is 7.35, and bio-concentration factor (LogBCF) is 4.30, shows that TBC is easy to enrichment in vivo; TBC reveals obvious bioconcentration at Different Nutrition level organism intensive amount change list, and toxic and endocrine disrupting to organism.In addition, bioconcentration and air free-radical oxidn transformation period result display TBC have the persistence similar with POPs, long range propagation and can the physicochemical property such as bioconcentration, and are enriched in cerebral tissue by hemato encephalic barrier.The long-distance migration characteristic of current TBC is unknown, but from its lower saturated vapor pressure (1.57 × 10
-13and higher octanol Pa)-air partition ratio (LogKoa=23.7) is analyzed, TBC can be adsorbed on particulate matter surface and with air-flow diffusion mobility.Although the production and application of present stage TBC is not applied to any restriction, it is included in, as the identification of a kind of high priority chemical by " Canadian environmental threat ecological material (LowerEcologicalConcern) screens register ".
At present to three (2,3-dibromopropyl) detection means of isocyanuric acid ester is mainly the instrument such as gas-chromatography and high performance liquid chromatography detection method, although these methods accurately and reliably, there is very high requirement to the pretreatment process of sample and the professional of operator.Just because of the process of these methods is complicated, consuming time, instrument price is expensive and be not suitable for promoting the use of, be also unfavorable in environmental pollution accident field quick detection.For overcoming these shortcomings, seek the main direction of studying that a kind of quick, easy, sensitive and economical and practical analytical procedure just becomes environmental monitoring field.
The immunoassay (Immunoassay, IA) that the sixties in 20th century grows up is based on antigen and the specificity of antibody, the analytical technology of reversibility association reaction.Immunoassay has the unrivaled selectivity of conventional physical and chemical analysis technology and high sensitivity, is applicable to very much the analysis of trace components in complex dielectrics.Therefore the high specificity, the advantage such as highly sensitive, method is quick and easy, analysis throughput is large, testing cost is low that have of immunoassay, makes these class methods can meet simply, detects the requirement of persistence organic pollutant fast, delicately.Within 1971, Engvail, VanWeerman etc. report the solid-phase immunoassay technology detecting micro substance in body fluid, i.e. enzyme linked immunosorbent assay analysis method (enzyme-linkedimmunosorbentassay, ELISA).At present, ELISA method has become integral part important in immune analysis method.ELISA method be by Ag-Ab between immune response and the efficient catalytic characteristic of enzyme organically combine and a kind of immune analysis method grown up.Wherein, the Avidin-Biotin enzyme linked immunosorbent assay analysis method based on Avidin-Biotin signal amplifying system, with biotin labelled antibodies (antigen), and replaces enzyme labelled antibody in ELISA method with enzyme mark avidin.Avidin is a kind of alkaline glycoprotein in Protalbinic acid, and molecular weight is about 68kDa.An avidin molecule is made up of 4 subunits, each subunit can with a biotin molecule (molecular weight is 244) specific binding.By force, its avidity is more much bigger than antigen antibody reaction, and affinity costant is up to 1015M for vitamin H and avidin binding specificity
-1.Because an avidin can be combined with 4 biotin molecules, therefore can improve by the quantity of solid phase desmoenzyme in the detection, and then improve the sensitivity of detection method
Avidin-Biotin enzyme linked immunosorbent assay analysis method is not still had to detect the relevant report of three (2,3-dibromopropyl) isocyanuric acid ester at present.
Summary of the invention
For defect of the prior art, the object of the invention is to provide first a kind of step simple, speed is fast, three (2,3-dibromopropyl) the isocyanuric acid ester haptens that productive rate is high and preparation thereof, purposes.
Research mechanism of the present invention is: for three (2,3-dibromopropyl) foundation of isocyanuric acid ester immunologic surveillance method, the haptens that high purity is suitable is that preparation has the basic of highly sensitive and specific immunogens and high-titer antibody, set up the key of three (2,3-dibromopropyl) isocyanuric acid ester immunologic detection method especially.Therefore, of the present invention three (2,3-dibromopropyl) the haptenic preparation of isocyanuric acid ester be adopt immunization method measure three (2,3-dibromopropyl) basis of isocyanuric acid ester, for the monitoring method setting up Polybrominated biphenyl monomer, there is important using value and theoretical significance.
The object of the invention is to be achieved through the following technical solutions:
First aspect, the present invention relates to a kind of three (2,3-dibromopropyl) isocyanuric acid ester hapten, its structural formula is such as formula shown in (I):
described formula I (three (2,3-dibromopropyl) isocyanuric acid ester hapten) is faint yellow acicular crystals, molecular formula: C
9h
10br
4n
3o
3, molecular weight 527.78; Fusing point: 128 ~ 131 DEG C.
Second aspect, the present invention relates to the haptenic preparation method of a kind of three (2,3-dibromopropyl) isocyanuric acid ester, described method comprises the steps:
S1, under non-proton organic solvent existent condition, 2,4,6-tri-(allyloxy)-1,3,5-triazines
with cupric chloride back flow reaction, generate diallyl isocyanuric acid ester;
S2, under methylene dichloride existent condition, diallyl isocyanuric acid ester and bromine back flow reaction, generate two (2, the 3-dibromopropyl) isocyanuric acid ester with secondary amino group active group, i.e. described three (2,3-dibromopropyl) isocyanuric acid ester hapten; The amino group of this introducing can with carrier protein couplet.
Preferably, step S1 is specially: be dissolved in non-proton organic solvent by three (2,3-dibromopropyl) isocyanuric acid esters and cupric chloride, obtain solution A, heat up gradually, keeps constant temperature back flow reaction subsequently, till raw material point disappears.
Preferably, step S2 is specially: diallyl isocyanuric acid ester is dissolved in non-proton organic solvent environment, then dropwise adds bromine and heats up gradually, keeps constant temperature back flow reaction subsequently, till raw material point disappears.
Its chemical equation is as follows:
Preferably, the mol ratio of described 2,4,6-tri-(allyloxy)-1,3,5-triazines, water, cupric chloride is 1: 1: 45 ~ 50; The mol ratio of described diallyl isocyanuric acid ester and bromine is 1: 4 ~ 5.
Ideally the mol ratio of three (2,3-dibromopropyl) isocyanuric acid ester, water, cupric chloride is 1: 1: 45, but carries out smoothly to react, and general cupric chloride can be more excessive, but can not exceed 1.5 times of desired quantity; Ideally 2,4,6-tri-(allyloxy)-1,3, the mol ratio that 5-triazine and cupric chloride react haptens intermediate and the bromine generated is 1: 4, for guaranteeing that reaction is carried out smoothly to the right, bromine is excessive, but can not exceed 5 times that 1.25 times of desirable molar weight are haptens intermediate molar weight.
Preferably, in step S1, the temperature of described back flow reaction is 95 ~ 100 DEG C, and the reaction times is 5 ~ 6 hours; In step S2, the temperature of described back flow reaction is 50 ~ 60 DEG C, and the reaction times is 1 ~ 2 hour.This temperature is more conducive to the carrying out reacted.
Preferably, the non-proton organic solvent in step S1, step S2 is independently selected from toluene, methylene dichloride or acetone.
Preferably, step S1 also comprises back flow reaction and terminates methylene dichloride and the sherwood oil recrystallization that rear volume ratio is 2: 1, and washing, obtains the step of the diallyl isocyanuric acid ester after purifying.
Preferably, step S2 also comprises back flow reaction and terminates to use saturated Na afterwards
2s
2o
3solution, with termination reaction, adds methylene dichloride in reaction solution, and be separated and dry organic phase, by dried organic phase desolventizing, chromatography column separation and purification, obtains the haptenic step of (2, the 3-dibromopropyl) isocyanuric acid ester of three after purifying.
Specifically, comprise the following steps:
B, reaction terminate to use saturated Na afterwards
2s
2o
3solution is with termination reaction;
C, in reaction solution, add methylene dichloride, be separated and successively wash with water, saturated NaCl solution repeatedly washs, then uses anhydrous Na
2sO
4dry organic phase is to remove unnecessary moisture;
D, by dried organic phase desolventizing, cross chromatography column separation and purification, finally obtain the faint yellow acicular crystals after purifying three (2,3-dibromopropyl) isocyanuric acid ester hapten two (2,3-dibromopropyl) isocyanuric acid ester, i.e. formula I.
Preferably, in step B, described saturated Na
2s
2o
3the phenomenon of solution termination reaction is that reaction system liquid redness disappears; Be conducive to termination reaction like this, also prevent the generation of reversed reaction.
Preferably, the thin-layer chromatography developping agent of described chromatography column separation and purification adopts volume ratio to be the ethyl acetate of 1: 1 and the mixing solutions of normal hexane composition.Haptens of the present invention is organic phase material, and the present invention surprisingly finds to adopt this developping agent to be more conducive to haptenic purifying, makes the haptens purity of acquisition higher.
The third aspect, the present invention relates to a kind of three (2,3-dibromopropyl) isocyanuric acid ester hapten intermediate, its structural formula is such as formula shown in (II):
Fourth aspect, the present invention relates to a kind of three (2,3-dibromopropyl) the artificial holoantigen of isocyanuric acid ester, described artificial holoantigen is former is prepared by three (2,3-dibromopropyl) as claimed in claim 1 isocyanuric acid ester hapten by glutaraldehyde method or carbodlimide method or diazotization method and protein molecule coupling and obtained.
Preferably, described artificial holoantigen comprises three (2,3-dibromopropyl) isocyanuric acid ester artificial immunogen, prepared by described three (2,3-dibromopropyl) isocyanuric acid ester hapten by glutaraldehyde method or carbodlimide method or diazotization method and bovine serum albumin coupling and obtain.
The structural formula of this artificial immunogen TBC-BSA is:
(glutaraldehyde method).This artificial immunogen immunize New Zealand White Rabbit, by the immunne response in organism and then the specific antibody preparing anti-three (2,3-dibromopropyl) isocyanuric acid ester
Preferably, described artificial holoantigen comprises three (2,3-dibromopropyl) the artificial coating antigen of isocyanuric acid ester, prepared by described three (2,3-dibromopropyl) isocyanuric acid ester hapten by glutaraldehyde method or carbodlimide method or diazotization method and oralbumin coupling and obtain.
The structural formula of this artificial coating antigen TBC-OVA is:
(carbodlimide method).This artificial coating antigen and three (2,3-dibromopropyl) isocyanuric acid ester can with the reaction resisting three (2,3-dibromopropyl) isocyanuric acid ester specific antibody immunoglobulin IgG generation specific recognition.
5th aspect, the present invention relates to a kind of three (2,3-dibromopropyl) isocyanuric acid ester hapten for the purposes in the detection of trace brominated flame-retardant three (2,3-dibromopropyl) isocyanuric acid ester.
Preferably, described detection comprises described three (2,3-dibromopropyl) isocyanuric acid ester hapten prepares the artificial holoantigen of three (2,3-dibromopropyl) isocyanuric acid esters by glutaraldehyde method or carbodlimide method or diazotization method and carrier protein couplet.
The present invention relates to three (2,3-dibromopropyl) isocyanuric acid ester hapten and preparation method thereof, purposes; Due to three (2,3-dibromopropyl) isocyanuric acid ester is small-molecule substance, only there is reactionogenicity and there is no immunogenicity, and this molecule there is no the functional group such as amino, carboxyl that directly can be combined with protein molecule, therefore need to make its molecule to be brought an amino to three (2,3-dibromopropyl) isocyanuric acid ester molecule derivatize.In order to the realization of immune analysis method, by glutaraldehyde method or carbodlimide method or diazotization method, this carboxylic haptens and protein molecule coupling are prepared artificial holoantigen (comprising artificial immunogen and artificial coating antigen), wherein artificial immunogen immunize New Zealand White Rabbit, by the immunne response in organism and then prepare anti-three (2, 3-dibromopropyl) specific antibody of isocyanuric acid ester, and artificial coating antigen and three (2, 3-dibromopropyl) isocyanuric acid ester can with anti-three (2, 3-dibromopropyl) reaction of isocyanuric acid ester specific antibody immunoglobulin IgG generation specific recognition, i.e. artificial coating antigen and three (2, 3-dibromopropyl) isocyanuric acid ester is the relation of direct competitive, thus set up immune analysis method for water body, soil, the environmental samples such as air and electronic product, textile printing and dyeing product, trace brominated flame-retardant three (2 in the products such as building decoration, 3-dibromopropyl) detection of isocyanuric acid ester.
Compared with prior art, the present invention has following beneficial effect:
1) haptens is practical: the preparation of three (2,3-dibromopropyl) isocyanuric acid ester hapten and antibody preparation have important practical value and realistic meaning.This haptens remains three (2,3-dibromopropyl) structure of isocyanuric acid ester, recycling glutaraldehyde method or carbodlimide method or diazotization method coupling protein matter makes it have for three (2,3-dibromopropyl) antigenic determinant of isocyanuric acid ester, can successfully prepare three (2,3-dibromopropyl) isocyanuric acid ester artificial immunogen TBC-BSA or three (2,3-dibromopropyl) the artificial coating antigen TBC-OVA of isocyanuric acid ester, for preparation specificity is good, the high antibody and set up immuno-PCR biobarcode approach and provide guarantee of tiring.
2) haptens good stability: the present invention has prepared three (2 first, 3-dibromopropyl) isocyanuric acid ester hapten, and three (2 of the synthesis of this method, 3-dibromopropyl) isocyanuric acid ester hapten structure has good stability, can preserve for many years under room temperature state, lose efficacy hardly.
3) haptens technology of preparing is simple and feasible: haptenic whole preparation process is without the need to special plant and instrument, with low cost, and preparation speed is fast, and productive rate is high, easy commercial scale production.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1 is the nmr spectrum of three (2,3-dibromopropyl) isocyanuric acid ester hapten intermediate;
Fig. 2 is the haptenic nmr spectrum of three (2,3-dibromopropyl) isocyanuric acid esters;
Fig. 3 is the haptenic infrared spectrogram of three (2,3-dibromopropyl) isocyanuric acid esters;
Fig. 4 is the ultraviolet spectrogram of three (2,3-dibromopropyl) isocyanuric acid ester hapten TBC-hapten, carrier proteins BSA and artificial holoantigen TBC-BSA;
Fig. 5 is the ultraviolet spectrogram of three (2,3-dibromopropyl) isocyanuric acid ester hapten TBC-hapten, carrier proteins OVA and artificial holoantigen TBC-OVA;
Fig. 6 is the amplification curve that indirect competitive detects three (2,3-dibromopropyl) isocyanuric acid ester immuno-PCR biobarcode approach;
Fig. 7 is the standard working curve that indirect competition immuno-PCR biobarcode approach detects three (2,3-dibromopropyl) isocyanuric acid ester.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
the Preparation and characterization of embodiment 1, three (2,3-dibromopropyl) isocyanuric acid ester hapten intermediate
(1) preparation of TBC haptens intermediate
Take 12.46g (0.05mol) 2,4,6-tri-(allyloxy)-1,3,5-triazines and 0.9g (0.05mol) water and 0.38g (2.25mmol) CuCl
22H
2o, joins after being dissolved in toluene in round-bottomed flask, is warming up to 95 DEG C of reflux 6 hours gradually, detects (R till raw material point disappears until thin plate chromatography (TLC)
f=0.2; Developping agent, normal hexane: ethyl acetate=5: 1); Be cooled to room temperature after reaction terminates, reaction solution is transferred to underpressure distillation in Rotary Evaporators and removes desolventizing, then use methylene dichloride and sherwood oil (V: V=2: 1) recrystallization, suction filtration obtains shallow green powder shape solid.Subsequently by dilute hydrochloric acid repeated washing green solid until color becomes white, then be washed with distilled water to weakly acidic pH, obtain diallyl isocyanuric acid ester and TBC haptens intermediate.Diallyl isocyanuric acid ester molecular formula: C
9h
11n
3o
3; Molecular weight: 209.2; Productive rate: 63.6%; Fusing point: 146-148 DEG C, purity 97%.
(2) sign of TBC haptens intermediate
The TBC haptens intermediate obtained, its structure is by detection means Analysis and Identification such as nucleus magnetic resonance, and as shown in Figure 1, its feature is as follows for the hydrogen nuclear magnetic resonance spectrogram of TBC haptens intermediate:
1hNMR (CDCl
3) δ (ppm): 8.44 (the non-phenyl ring of 1HArH), 5.89 (1H-C=C-H conjugation), 5.23-5.34 (2H-C=CH
2), 4.45,4.47 (2HAr-CH
2-R).Nuclear-magnetism characterization result shows, Success in Experiment has prepared TBC haptens intermediate.
the Preparation and characterization of embodiment 2, three (2,3-dibromopropyl) isocyanuric acid ester hapten intermediate
(1) preparation of TBC haptens intermediate
Take 12.46g (0.05mol) 2,4,6-tri-(allyloxy)-1,3,5-triazines and 0.9g (0.05mol) water and 0.42g (2.5mmol) CuCl
22H
2o, joins after being dissolved in toluene in round-bottomed flask, is warming up to 95 DEG C of reflux 5 hours gradually, detects (R till raw material point disappears until thin plate chromatography (TLC)
f=0.2; Developping agent, normal hexane: ethyl acetate=5: 1); Be cooled to room temperature after reaction terminates, reaction solution is transferred to underpressure distillation in Rotary Evaporators and removes desolventizing, then use methylene dichloride and sherwood oil (V: V=2: 1) recrystallization, suction filtration obtains shallow green powder shape solid.Subsequently by dilute hydrochloric acid repeated washing green solid until color becomes white, then be washed with distilled water to weakly acidic pH, obtain diallyl isocyanuric acid ester and TBC haptens intermediate.Diallyl isocyanuric acid ester molecular formula: molecular formula: C
9h
11n
3o
3; Molecular weight: 209.2; Productive rate: 63.6%; Fusing point: 146-148 DEG C, purity 97%.
(2) sign of TBC haptens intermediate
The TBC haptens intermediate obtained, its structure is by detection means Analysis and Identification such as nucleus magnetic resonance, and the hydrogen nuclear magnetic resonance spectrogram feature of TBC haptens intermediate is as follows:
1hNMR (CDCl
3) δ (ppm): 8.44 (the non-phenyl ring of 1HArH), 5.89 (1H-C=C-H conjugation), 5.23-5.34 (2H-C=CH
2), 4.45,4.47 (2HAr-CH
2-R).Nuclear-magnetism characterization result shows, Success in Experiment has prepared TBC haptens intermediate.
the haptenic Preparation and characterization of embodiment 3, three (2,3-dibromopropyl) isocyanuric acid ester
(1) the haptenic preparation of TBC
Take 0.5g (0.9448mmol) TBC haptens intermediate (diallyl isocyanuric acid ester) and be dissolved in 20mLCH
2cl
2in; Under heating 50 DEG C of states, dropwise add 120 μ L (3.78mmol) bromines in aforesaid liquid, keep back flow reaction 2 hours, thin plate chromatography (TLC) monitoring reaction is until react completely; After reaction solution is cooled to room temperature, in reaction system, add saturated Na
25
2o
3solution, until reaction system liquid redness disappears; With 100mL water and 150mL (50mL × 3 time) CH
2cl
2re-extract reaction solution, subsequently, is separated and retains organic phase, and repeatedly washing by saturated NaCl solution, then using anhydrous Na
2sO
4dry organic phase, to remove unnecessary moisture, obtains two (2,3-dibromopropyl) isocyanuric acid ester crude product.Mixed solution (V/V=1: the 1) recrystallization of crude product ethyl acetate and normal hexane, obtains two (2,3-dibromopropyl) isocyanuric acid ester and TBC haptens.Three (2,3-dibromopropyl) isocyanuric acid ester hapten molecule formula: C
9h
10br
4n
3o
3; Molecular weight 527.78; Productive rate 47.1%; Fusing point: 128 ~ 131 DEG C, purity 98%.
(2) the haptenic sign of TBC
The TBC haptens obtained, its structure is by detection means Analysis and Identification such as nucleus magnetic resonance, infrared spectra, UV spectrum, and as shown in Figure 2, its feature is as follows for the hydrogen nuclear magnetic resonance spectrogram of TBC haptens intermediate:
1hNMR (CDCl
3) δ (ppm): 9.22 (1HN-H), 4.42 (1HR-CH-Br), 3.77,3.88 (2H-CH
2-Br), 1.44,1.28 (2H-CH
2-).Nuclear-magnetism has obvious-NH group characteristic peak in characterizing, and result shows: Success in Experiment has prepared TBC haptens.
The haptenic diffuse reflectance infrared spectroscopy of TBC is as follows: IR (KBr) v/cm
-1: 1671.98 (C=O stretches), 1430.92 (O-H in-plane bendings); 3320.82 (parahelium N-H stretches), 1295.93,1315.21 (C-N fragrance is flexible); 2989.12 (C-H stretches), 1648.84 ~ 1432.85 (C=C phenyl ring skeletal vibrations), 910 ~ 665 (C-H out-of-plane bendings), 621.08 (C-Br stretches).Find out there are the group charateristic avsorption bands such as obvious-COOH ,-NH, phenyl ring, C-Br in the infrared spectra of TBC hapten molecule from the infared spectrum interpretation of result of Fig. 3, prove in the TBC hapten molecule prepared containing amido functional group.
the haptenic Preparation and characterization of embodiment 4, three (2,3-dibromopropyl) isocyanuric acid ester
(1) the haptenic preparation of TBC
Take 0.5g (0.9448mmol) TBC haptens intermediate (diallyl isocyanuric acid ester) and be dissolved in 20mLCH
2cl
2in; Under heating 60 DEG C of states, dropwise add 132 μ L (4.16mmol) bromines in aforesaid liquid, keep back flow reaction 1 hour, thin plate chromatography (TLC) monitoring reaction is until react completely; After reaction solution is cooled to room temperature, in reaction system, add saturated Na
25
2o
3solution, until reaction system liquid redness disappears; With 100mL water and 150mL (50mL × 3 time) CH
2cl
2re-extract reaction solution, subsequently, is separated and retains organic phase, and repeatedly washing by saturated NaCl solution, then using anhydrous Na
2sO
4dry organic phase, to remove unnecessary moisture, obtains two (2,3-dibromopropyl) isocyanuric acid ester crude product.Mixed solution (V/V=1: the 1) recrystallization of crude product ethyl acetate and normal hexane, obtains two (2,3-dibromopropyl) isocyanuric acid ester and TBC haptens.Three (2,3-dibromopropyl) isocyanuric acid ester hapten molecule formula: C
9h
10br
4n
3o
3; Molecular weight 527.78; Productive rate 47.1%; Fusing point: 128 ~ 131 DEG C, purity 98%.
(2) the haptenic sign of TBC
The TBC haptens obtained, its structure is by detection means Analysis and Identification such as nucleus magnetic resonance, infrared spectra, UV spectrum, and the hydrogen nuclear magnetic resonance spectrum signature of TBC haptens intermediate is as follows:
1hNMR (CDCl
3) δ (ppm): 9.22 (1HN-H), 4.42 (1HR-CH-Br), 3.77,3.88 (2H-CH
2-Br), 1.44,1.28 (2H-CH
2-).Nuclear-magnetism has obvious-NH group characteristic peak in characterizing, and result shows: Success in Experiment has prepared TBC haptens.
The haptenic diffuse reflectance infrared spectroscopy of TBC is as follows: IR (KBr) v/cm
-1: 1671.98 (C=O stretches), 1430.92 (O-H in-plane bendings); 3320.82 (parahelium N-H stretches), 1295.93,1315.21 (C-N fragrance is flexible); 2989.12 (C-H stretches), 1648.84 ~ 1432.85 (C=C phenyl ring skeletal vibrations), 910 ~ 665 (C-H out-of-plane bendings), 621.08 (C-Br stretches).Find out there are the group charateristic avsorption bands such as obvious-COOH ,-NH, phenyl ring, C-Br in the infrared spectra of TBC hapten molecule from the analysis of infared spectrum data results, prove in the TBC hapten molecule prepared containing amido functional group.
the preparation of the artificial holoantigen of embodiment 5, three (2,3-dibromopropyl) isocyanuric acid ester
Three (2,3-dibromopropyl) isocyanuric acid ester hapten prepared by the present invention, is mainly used in the immunodetection of three (2,3-dibromopropyl) isocyanuric acid ester in environment.One of its main application, can be used for exactly directly and protein macromolecule coupling, prepare the immunogen for immune animal, and then prepare corresponding mono-clonal or polyclonal antibody.Set up the method for immunity of three (2,3-dibromopropyl) isocyanuric acid ester on this basis.Following applicating example is used as below with regard to haptenic the making of three (2,3-dibromopropyl) isocyanuric acid ester:
Glutaraldehyde method is adopted to prepare TBC-BSA immunogen, concrete synthesis step: to take 0.1434g (0.5mmol) TBC haptens, be dissolved in 1mLDMF; Taking 120mgBSA is dissolved in 10mL0.01MpH7.40PBS damping fluid; Under 4 DEG C of magnetic agitation, dropwise by TBC haptens solution instillation BSA solution, drip and terminate to add 200 μ L25% glutaraldehyde in backward reaction system, 4 DEG C of low-temperature magnetic stir 24h; After reaction terminates, low-temperature centrifugation separation of supernatant, loads supernatant liquor in dialysis tubing, with the PBS dialysis 3d of 0.01MpH7.40, changes water every day 3 times, obtains TBC immunogen TBC-BSA after centrifugation precipitation.Immunogen after ultraviolet-visible spectrophotometer qualification, in a small amount packing ,-20 DEG C of freezen protective.
TBC-OVA coating antigen is prepared, concrete synthesis step: take 0.2645g (0.5mmol) TBC haptens and be dissolved in 1mLDMF by carbodlimide method; Take 0.09585g (0.5mmol) 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) and 0.1125gOVA is dissolved in the NaAc-HAc damping fluid of 0.01MpH5.0 respectively; In OVA solution, slowly drip TBC haptens and EDC solution, 6h is stirred in 4 DEG C of ice bath shadings, then in mixed solution, add other 10mgEDC solution, continues 4 DEG C of ice bath shadings and stirs 24h.After reaction terminates, the centrifugal 10-15min separation of supernatant of 3000rpm, supernatant liquor 0.01MpH7.4PBS damping fluid dialysis 3d, changes water every day 3 times.After dialysis, centrifugation precipitates, and obtains TBC coating antigen TBC-OVA.Coating antigen after ultraviolet-visible spectrophotometer qualification, in a small amount packing ,-20 DEG C of freezen protective.
Conjugate is after ultraviolet-visible pectrophotometer scanning qualification, and as can be seen from the TBC haptens of Fig. 4 and Fig. 5, carrier proteins and artificial holoantigen ultraviolet spectrogram, the haptenic charateristic avsorption band of TBC is positioned at 281nm; The charateristic avsorption band of carrier proteins BSA is positioned at 227nm and 273nm place; The charateristic avsorption band of carrier proteins OVA appears at 219nm, 224nm, 268nm place.Not only comprise the characteristic peak of BSA and OVA in the UV spectrum of conjugate TBC-BSA and TBC-OVA respectively, also occur new absorption peak in 256 and 253nm place.TBC-BSA and TBC-OVA haptens charateristic avsorption band there occurs blue shift, and this may be subject to carrier proteins caused by the impact of about 270nm absorption peak.The appearance of absorption peak blue-shifted phenomenon, illustrates that above information shows TBC-BSA and TBC-OVA coupling success.
These holoantigens both may be used for immune animal, and the immune response produced by animal obtains the mono-clonal that can be used for doing immunoassay or polyclonal antibody, can use again as Immune competition object.
embodiment 6, indirect competition immuno-PCR biobarcode approach detect three (2,3-dibromopropyl) isocyanuric acid ester
In order to strengthen the adsorptivity of PCR pipe, the glutaraldehyde with 0.8% carries out pre-treatment to PCR pipe, and every hole adds 20 μ L glutaraldehyde solutions, and incubation 5 hours at 37 DEG C, then uses milli-Q water 3 times, dry for subsequent use; Be buffered liquid (0.05MpH9.60 carbonate buffer solution) with bag and coating antigen TBC-OVA is diluted to proper concn, join and carry out bag quilt in the PCR pipe of glutaraldehyde process, every hole 20 μ L, wraps at 0 ~ 4 DEG C and is spent the night; Outwell coating antigen, every hole adds 200 μ L washingss (0.01MpH7.40PBST) and washs, and vibrates after 3 minutes and dries, add 200 μ L confining liquids (phosphate buffer soln containing 1%OVA), incubation 1 hour at 37 DEG C after washing three times; Wash plate three times, anti-TBC polyclonal antibody is diluted to proper concn, every hole adds 10 μ L antibody and 10 μ LTBC small molecules, adds 20 μ LPBS in blank control wells, incubation 1 hour at 37 DEG C; Wash plate three times and dry, with 0.01MpH7.40PBS, gold nano grain bioprobe being diluted to suitable multiple, every hole adds 20 μ L, incubation 1 hour at 37 DEG C; Use elutriant repeated washing five times after outwelling solution, then use ultrapure water repeated washing five times; Finally add amplimer and fluorescent substance, adopt the amplification system of 20 μ L, every hole adds 10 μ LPCR test kit mixture, and upstream and downstream primer respectively adds 0.25 μ L, ultrapure water 9.5 μ L; When adding pcr amplification reagent, in Fluorescence PCR instrument, carry out (the pcr amplification program: 95 DEG C of denaturation 5min that increases; 95 DEG C of 20s, 57 DEG C of 20s, 72 DEG C of 20s, carry out 35 circulations with this; 72 DEG C extend 3min).The Ct value measured and the amount of determined antigen are inversely proportional to.Antigen and corresponding Ct value according to adding concentration known make typical curve, thus can obtain the concentration of TBC in corresponding testing sample.The typical curve of its amplification curve measured and foundation as shown in Figure 6 and Figure 7.
In this experiment, barcode DNA used takes from a section in PUC19 plasmid DNA.According to design of primers principle, use the primer that primerpremier5.0 software design is suitable ', upstream primer 5 '-GAGGCGGTTTGCGTATTG-3 ' and downstream primer 5 '-AGCGAGGAAGCGGAAGAG-3 ', the preparation synthesis of student on commission's work biotechnology (Shanghai) limited-liability company.
When TBC concentration is at 0.1pg/L-100pg/L, linear dependence is better, and linear equation is Ct=1.071IgC+14.3 (R
2=0.974, n=5), LOD=0.97pg/L.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (10)
1. one kind three (2,3-dibromopropyl) isocyanuric acid ester hapten, is characterized in that, its structural formula is such as formula shown in (I):
2. the haptenic preparation method of three (2,3-dibromopropyl) isocyanuric acid ester as claimed in claim 1, it is characterized in that, described method comprises the steps:
S1, under non-proton organic solvent existent condition, 2,4,6-tri-(allyloxy)-1,3,5-triazines
with cupric chloride back flow reaction, generate diallyl isocyanuric acid ester;
S2, under non-proton organic solvent existent condition, diallyl isocyanuric acid ester and bromine back flow reaction, generate described three (2,3-dibromopropyl) isocyanuric acid ester hapten.
3. the haptenic preparation method of three (2,3-dibromopropyl) isocyanuric acid ester according to claim 2, is characterized in that, described 2,4,6-tri-(allyloxys)-1, the mol ratio of 3,5-triazine, water, cupric chloride is 1: 1: 45 ~ 50; The mol ratio of described diallyl isocyanuric acid ester and bromine is 1: 4 ~ 5.
4. the haptenic preparation method of three (2,3-dibromopropyl) isocyanuric acid ester according to claim 2, is characterized in that, in step S1, the temperature of described back flow reaction is 95 ~ 100 DEG C, and the reaction times is 5 ~ 6 hours; In step S2, the temperature of described back flow reaction is 50 ~ 60 DEG C, and the reaction times is 1 ~ 2 hour.
5. the haptenic preparation method of three (2,3-dibromopropyl) isocyanuric acid ester according to claim 2, it is characterized in that, the non-proton organic solvent in step S1, step S2 is independently selected from toluene, methylene dichloride or acetone.
6. according to claim 2 three (2,3-dibromopropyl) the haptenic preparation method of isocyanuric acid ester, it is characterized in that, the back flow reaction that also comprises step S1 terminates methylene dichloride and the sherwood oil recrystallization that rear volume ratio is 2: 1, washing, obtains the step of the diallyl isocyanuric acid ester after purifying.
7. the haptenic preparation method of three (2,3-dibromopropyl) isocyanuric acid ester according to claim 2, is characterized in that, step S2 also comprises back flow reaction to be terminated to use saturated Na afterwards
2s
2o
3solution, with termination reaction, adds methylene dichloride in reaction solution, and be separated and dry organic phase, by dried organic phase desolventizing, chromatography column separation and purification, obtains the haptenic step of (2, the 3-dibromopropyl) isocyanuric acid ester of three after purifying.
8. one kind three (2,3-dibromopropyl) isocyanuric acid ester hapten intermediate, it is characterized in that, its structural formula is such as formula shown in (II):
9. one kind three (2,3-dibromopropyl) the artificial holoantigen of isocyanuric acid ester, it is characterized in that, described artificial holoantigen is prepared by three (2,3-dibromopropyl) as claimed in claim 1 isocyanuric acid ester hapten by glutaraldehyde method or carbodlimide method or diazotization method and protein molecule coupling and obtained.
10. the purposes of three (2,3-dibromopropyl) as claimed in claim 1 isocyanuric acid ester hapten in detecting for trace brominated flame-retardant three (2,3-dibromopropyl) isocyanuric acid ester.
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