CN105085300A - Plasticizer di(2-ethyl)hexyl phthalate hapten and preparation method thereof - Google Patents
Plasticizer di(2-ethyl)hexyl phthalate hapten and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a plasticizer di(2-ethyl)hexyl phthalate hapten and a preparation method thereof. The structural formula of the di(2-ethyl)hexyl phthalate hapten is shown in the specification. The DEHP hapten containing an amino active group is prepared by taking 4-nitrophthalic acid as a raw material through two-step reaction. The di(2-ethyl)hexyl phthalate hapten is simple and convenient in preparation method, good in stability, low in cost and easy for industrialized production; the di(2-ethyl)hexyl phthalate hapten is coupled with a carrier protein through a glutaraldehyde process, a diazotization process or a carbodiimide process to prepare a di(2-ethyl)hexyl phthalate artificial holoantigen; guarantee is provided for preparing an antibody with good specificity and high titer and building a novel immunoassay method.
Description
Technical field
The present invention relates to a kind of haptens and preparation method thereof, especially relate to own ester hapten of a kind of fluidizer phthalic acid two (2-ethyl) and preparation method thereof.
Background technology
Phthalate fluidizer (being abbreviated as PAEs), also known as phthalate, is the general designation of the important derivatives of the ester that a class is formed with alcohol generation Fischer (Fischer) esterification containing 4-15 carbon by phthalic acid.This kind of material has specific characteristics smell, and toxicity is comparatively large, and generally state is thick liquid; Under liquid condition, temperature range is wide, and mobility is large, and volatility is low, water insoluble, is soluble in most of organic solvent.This kind of substance use is extensive, it not only can be used as the production starting material of nearly thousand kinds of products such as packaging material for food, toy, farm chemical carrier, wormer, lubricant, vinyl flooring and wallpaper, defoaming agents, sanitising agent, medical material artificial organs, blood bag, syringe and sebific ducts such as (as:) heart valve prosthesises and personal-care supplies (mainly containing makeup, fragrance product, nail varnish, hair spray, perfumed soap and shampoo), and also Chang Zuowei plastic plasticizer is used to transformation plastics performance.Be prevalent in the natures such as air, water body, soil, biology and even human body and human environment due to PAEs, and all generally can detect in each major industrial country's environment of the whole world, so they oneself become the most general global ubiquitous pollutent, be also referred to as the 2nd class global " PCB pollutent " simultaneously.
Wherein, the own ester of phthalic acid two-(2-ethyl) (English name: Diethylhexylphthalate, vehicle economy HP), have another name called two (2-ethyl hexyl) ester of phthalandione, No. CAS is 117-81-7, and molecular formula is C
24h
38o
4, molecular weight is 390.56, colourless liquid, is soluble in ethanol, ether and mineral wet goods.The strong toxicity of this material, may cause the entanglement of child's sex, sexual organ short and small, can produce carcinogenic reaction to animal; Can add up in human body, can male reproductive function be endangered, impel female precocious puberty.This material widely uses mainly as the softening agent of plasthetics (as: soft PVC product, medication injections articles for use, toy etc.).In the last few years, detect that this substances content is higher, constitute a threat to HUMAN HEALTH in Taiwan Food, therefore, the content of DEHP in varieties of food items causes showing great attention to of society.
At present the instrument such as gas-chromatography and high performance liquid chromatography detection method is mainly to the detection means of phthalic acid two (2-ethyl) own ester, 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, Avidin-Biotin ELISA adsorption analysis method (BA-ELISA) based on Avidin-Biotin signal amplifying system, with biotin labelled antibodies (antigen), and replace 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 10 for vitamin H and avidin binding specificity
15m
-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 phthalic acid two (2-ethyl) own ester at present.For the foundation of the own ester immunologic surveillance method of phthalic acid two (2-ethyl), the haptens that high purity is suitable is that preparation has the basic of highly sensitive and specific immunogens and high-titer antibody, sets up the key of phthalic acid two (2-ethyl) own ester immunologic detection method especially.
Therefore, the preparation of phthalic acid two (2-ethyl) own ester hapten is the basis adopting immunization method to measure phthalic acid two (2-ethyl) own ester, for the monitoring method setting up Polybrominated biphenyl monomer, there is important using value and theoretical significance.
Summary of the invention
For defect of the prior art, the object of this invention is to provide own ester hapten of a kind of fluidizer phthalic acid two (2-ethyl) and preparation method thereof, that a kind of step is simple, speed is fast, phthalic acid two (2-ethyl) own ester hapten that productive rate is high and preparation method thereof.
The object of the invention is to be achieved through the following technical solutions:
First aspect, the invention provides the own ester hapten of a kind of phthalic acid two (2-ethyl), structural formula is such as formula shown in I:
Described formula I is yellow needles body, and fusing point is 34 ~ 36 DEG C.
Second aspect, the invention still further relates to the preparation method of the own ester hapten of a kind of described phthalic acid two (2-ethyl), described preparation method comprises the steps:
Haptens Intermediate Preparation: in strong acid environment, 4-nitrophthalic acid and isooctyl alcohol generation esterification, generate 4-nitrophthalic acid two (2-ethyl) own ester, be haptens intermediate;
Prepared by haptens: in non-proton organic solvent environment, described haptens intermediate and zinc powder, strong acid react, and obtain phthalic acid two (2-ethyl) own ester hapten; Also i.e. DEHP haptens, with amino active group, the amino group of introducing can with carrier protein couplet.
Preferably, in the step of described haptens Intermediate Preparation, described strong acid is the vitriol oil or concentrated hydrochloric acid.
Preferably, the step of described haptens Intermediate Preparation is specially: be dissolved in isooctyl alcohol solution by 4-nitrophthalic acid, be added dropwise to strong acid, the reaction system generation esterification of formation;
Described dropping strong acid solution is specially and dropwise drips, and be the routine operation of esterification, strong acid can not add fast, dropwise add, and prevents local release of heat from too much causing bulk temperature uneven, easily like this sets off an explosion; The degree of described generation esterification is till raw material point disappears.
Preferably, in the step of described haptens Intermediate Preparation, the mol ratio of 4-nitrophthalic acid, isooctyl alcohol, strong acid is 1:5 ~ 6:0.6 ~ 0.7.As shown in chemical reaction equation, ideally the mol ratio of 4-nitrophthalic acid, isooctyl alcohol is 1:2, but carry out to the right smoothly to react, the consumption of general isooctyl alcohol is more than 5 times of the molar weight of 4-nitrophthalic acid, but can not more than 6 of the molar weight of 4-nitrophthalic acid times; In addition, strong acid is as the catalyzer of esterification, and amount used is generally more than 0.6 times of raw material 4-nitrophthalic acid mole number, but is no more than 0.7 times of raw material 4-nitrophthalic acid mole number.
Preferably, in the step of described haptens Intermediate Preparation, the temperature of described reaction is 120 ~ 150 DEG C, and the time of described reaction is 5 ~ 6 hours; This temperature is more conducive to the carrying out of esterification, and detects with thin-layer chromatography, and constant temperature backflow can react completely for 5 ~ 6 hours.
Preferably, the step of described haptens Intermediate Preparation also comprises:
A1, remove the water that isooctyl alcohol that esterification does not occur in described reaction system and reaction generate, then remainder is poured in frozen water, precipitate is haptens crude intermediate; Be conducive to termination reaction like this, also prevent the generation of reversed reaction;
A2, be colourless by the washing of described haptens crude intermediate to washings, dry, obtain the haptens intermediate of purifying.
Preferably, in steps A 1, the method removing the water of isooctyl alcohol and the reaction generation that esterification does not occur in described reaction system adopts distillation; Describedly to pour into as pouring into while hot.
Preferably, in steps A 2, described washing specifically adopt massfraction be 10% Na
2cO
3solution; When washing is colourless to washings, its pH is 7.0 ~ 8.0; Described drying is specially and adopts anhydrous Na2SO4 dehydration dry; Obtain yellow oily liquid and 4-nitrophthalic acid two (2-ethyl) own ester by steps A 2, namely phthalic acid two (2-ethyl) own ester hapten intermediate, i.e. formula II compound, molecular formula is C
24h
37nO
6, molecular weight is 435.55.
Preferably, the step that prepared by described haptens is specially: by haptens intermediate in non-proton organic solvent environment; Then add zinc powder, mix; Be added dropwise to strong acid again, again add zinc powder, the reaction system isothermal reaction of formation;
Described dropping dropwise adds, and this operation belongs to carries out the routine operation of esterification, and strong acid can not add fast, dropwise add, and prevents local release of heat from too much causing bulk temperature uneven, easily like this sets off an explosion; Till described isothermal reaction continues to the disappearance of raw material point; Described zinc powder is pure zinc powder, i.e. chemical rank analytical pure, and traditional Chinese medicines Reagent Company produces.
Preferably, described non-proton organic solvent comprises benzene, toluene or acetone; Described strong acid is concentrated hydrochloric acid or the vitriol oil; Described mix into magnetic agitation mixing.
Preferably, the mol ratio of described haptens intermediate, strong acid, nonionic organic solvent and zinc powder is 1:(20 ~ 25): (550 ~ 580): (18 ~ 20), wherein nonionic organic solvent does not participate in reaction as matrix solution.As shown in chemical reaction equation, ideally the mol ratio of haptens intermediate 4-nitrophthalic acid two (2-ethyl) own ester, strong acid and zinc powder is 1:55:10, but carry out to the right smoothly to react, the consumption of general pure zinc powder is more than 18 times of raw material 4-nitrophthalic acid molar weight, but can not exceed 20 times of raw material 4-nitrophthalic acid molar weight; In addition, strong acid is as the strong oxidizer of reduction reaction, amount used is generally more than 20 times of raw material 4-nitrophthalic acid two (2-ethyl) own ester molar weight, but be no more than 25 times of raw material 4-nitrophthalic acid two (2-ethyl) own ester molar weight, and the mole number of aprotic organic solvent-benzene needs to be more than 20 times of strong oxidizer mole number, but be no more than 28 times of strong oxidizer mole number.
Preferably, the temperature of described isothermal reaction is 25 ~ 30 DEG C, and the time of described isothermal reaction is 10 ~ 12 hours.This temperature is more conducive to the carrying out reacted, and detects with thin-layer chromatography, and constant temperature backflow can react completely for 10 ~ 12 hours.
Preferably, described haptens is prepared further comprising the steps of:
After B1, described reaction terminate, cool described reaction system, adjustment pH is weakly alkaline;
B2, leave standstill, extract to obtain extraction liquid, wash described extraction liquid, dry;
Organic solvent constituent in B3, the dry rear extraction liquid of removing, obtains yellow solid;
B4, by gained yellow solid through silica gel column chromatography, then underpressure distillation, obtains phthalic acid two (2-ethyl) own ester hapten, namely 4-aminophthalic acid two (2-ethyl) own ester, i.e. formula I.
Preferably, in step B1, described weakly alkaline specifically refers to pH7.0 ~ 8.5; Described cooling is specifically pointed in described reaction system and is added cold water.
Preferably, in step B1, described adjustment pH adopts strong alkali solution, and described strong alkali solution is sodium hydroxide or potassium hydroxide.
Preferably, in step B2, described extraction adopts benzene.
Preferably, in step B2, the described standing time is 1h; Described washing is washing; Described drying specifically adopts anhydrous Na
2sO
4dehydrate.
Preferably, in step B4, described silica gel column chromatography elutriant used adopts volume ratio to be the acetic acid of 1:15 and the mixing solutions of normal hexane composition.Because haptens is organic phase material, the present invention surprisingly finds to adopt this developping agent (normal hexane: ethyl acetate=5:1) to be more conducive to haptenic purifying, makes the haptens purity of acquisition higher; Step B4 obtains yellow crystals and 4-aminophthalic acid two (2-ethyl) own ester, namely phthalic acid two (2-ethyl) own ester hapten, i.e. formula I.Phthalic acid two (2-ethyl) own ester hapten molecular formula: C
24h
39nO
4, molecular weight: 405.57; Fusing point: 34 ~ 36 DEG C.
Chemical equation prepared by above-mentioned haptens is as follows:
The third aspect, present invention also offers the purposes of the own ester hapten of a kind of described phthalic acid two (2-ethyl), specifically for the detection of trace fluidizer phthalic acid two (2-ethyl) own ester.
Fourth aspect, the invention provides the holoantigen of the own ester hapten of a kind of described phthalic acid two (2-ethyl), described holoantigen is prepared by the following method: by phthalic acid two (2-ethyl) own ester hapten by glutaraldehyde method or diazotization method or carbodlimide method and carrier protein couplet, to obtain final product.
Preferably, described carrier proteins is bovine serum albumin or oralbumin.
The holoantigen of the own ester hapten of described phthalic acid two (2-ethyl) is the own ester artificial immunogen DEHP-BS of phthalic acid two (2-ethyl)
or
The artificial coating antigen DEHP-OVA of phthalic acid two (2-ethyl) own ester
Wherein, BSA is bovine serum albumin, and OVA is oralbumin.
The present invention relates to own ester hapten of a kind of phthalic acid two (2-ethyl) and preparation method thereof, because phthalic acid two (2-ethyl) own 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 phthalic acid two (2-ethyl) own ester molecule derivatize.In order to the realization of immune analysis method, with glutaraldehyde method or diazotization method or carbodlimide 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 the specific antibody preparing the own ester of anti-phthalic acid two (2-ethyl), and artificial coating antigen and phthalic acid two (2-ethyl) own ester can with the reaction of anti-phthalic acid two (2-ethyl) own ester specific antibody immunoglobulin IgG generation specific recognition, namely artificial coating antigen and phthalic acid two (2-ethyl) own ester are the relations of direct competitive, thus set up immune analysis method for water body, soil, the detection of trace fluidizer phthalic acid two (2-ethyl) own ester in the environmental samples such as air and varieties of food items.
Compared with prior art, the present invention has following beneficial effect:
1, haptens is practical: the own ester hapten preparation of phthalic acid two (2-ethyl) and antibody preparation have important practical value and realistic meaning; This haptens remains the structure of phthalic acid two (2-ethyl) own ester, recycling glutaraldehyde method or diazotization method or carbodlimide method coupling protein matter make it have the antigenic determinant for phthalic acid two (2-ethyl) own ester, successfully can prepare phthalic acid two (2-ethyl) own ester artificial immunogen DEHP-BSA or the artificial coating antigen DEHP-OVA of phthalic acid two (2-ethyl) own ester, for preparation specificity is good, the high antibody and set up Novel immune analytical procedure and provide guarantee of tiring;
2, haptens good stability: the present invention has prepared phthalic acid two (2-ethyl) own ester hapten first, and phthalic acid two (2-ethyl) the own ester hapten structure of this method synthesis 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 infrared spectrogram of phthalic acid two (2-ethyl) own ester hapten intermediate;
Fig. 2 is the infrared spectra of phthalic acid two (2-ethyl) own ester hapten;
Fig. 3 is the nmr spectrum of phthalic acid two (2-ethyl) own ester hapten;
Fig. 4 is the ultraviolet spectrogram of phthalic acid two (2-ethyl) own ester hapten DEHP-hapten, carrier proteins BSA and artificial holoantigen DEHP-BSA;
Fig. 5 is the ultraviolet spectrogram of phthalic acid two (2-ethyl) own ester hapten DEHP-hapten, carrier proteins OVA and artificial holoantigen DEHP-OVA;
Fig. 6 is the standard working curve that indirect competition BA-ELISA immune analysis method detects phthalic acid two (2-ethyl) own 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 of embodiment 1, phthalic acid two (2-ethyl) own ester hapten intermediate
Getting clean volume is 100mL tri-mouthfuls of round-bottomed flasks, loads 10g (0.0475mol) 4-nitrophthalic acid, slowly add 44.6mL (0.2850mol) isooctyl alcohol subsequently, add the dense H of 1.65mL while stirring at the bottom of bottle
2sO
4(0.0304mol) catalysis, 120 DEG C of stirring and refluxing, reaction mixture drips to tlc silica gel plate (2.5cm × 8.0cm) so that whether inspection reaction completes; (developer: dissolve a small amount of potassium permanganate in acetone solvent till raw material point disappears is detected until thin plate chromatography (TLC); Developping agent, normal hexane: ethyl acetate=5:1); React after stirring and refluxing 6h;
The water of the distillation unreacted isooctyl alcohol of removing and generation; Now liquid is poured in frozen water while hot, leave standstill for some time layering after magnetic agitation 5min, by lower floor yellow oily liquid 10%Na
2cO
3solution washing to water layer is colourless (pH7.0-8.0); Finally, crude oil is dry with anhydrous sodium sulfate dehydration, obtains yellow oily liquid 4-nitrophthalic acid two (2-ethyl) own ester and DEHP haptens intermediate.4-nitrophthalic acid two (2-ethyl) own ester molecule formula: C
24h
37nO
6, molecular weight: 435.55; Productive rate: 92.8%; Purity 97%.
the preparation of embodiment 2, phthalic acid two (2-ethyl) own ester hapten intermediate
Getting clean volume is 100mL tri-mouthfuls of round-bottomed flasks, loads 10g (0.0475mol) 4-nitrophthalic acid, slowly add 37.2mL (0.2375mol) isooctyl alcohol subsequently, add the dense H of 1.55mL while stirring at the bottom of bottle
2sO
4(0.0285mol) catalysis, 150 DEG C of stirring and refluxing, reaction mixture drips to tlc silica gel plate (2.5cm × 8.0cm) so that whether inspection reaction completes; (developer: dissolve a small amount of potassium permanganate in acetone solvent till raw material point disappears is detected until thin plate chromatography (TLC); Developping agent, normal hexane: ethyl acetate=5:1); React after stirring and refluxing 6h;
The water of the distillation unreacted isooctyl alcohol of removing and generation; Now liquid is poured in frozen water while hot, leave standstill for some time layering after magnetic agitation 5min, by lower floor yellow oily liquid 10%Na
2cO
3solution washing to water layer is colourless (pH7.0-8.0); Finally, crude oil is dry with anhydrous sodium sulfate dehydration, obtains yellow oily liquid 4-nitrophthalic acid two (2-ethyl) own ester and DEHP haptens intermediate; 4-nitrophthalic acid two (2-ethyl) own ester molecule formula: C
24h
37nO
6, molecular weight: 435.55; Productive rate: 92.8%; Purity 97%.
the preparation of embodiment 3, phthalic acid two (2-ethyl) own ester hapten intermediate
Getting clean volume is 100mL tri-mouthfuls of round-bottomed flasks, loads 10g (0.0475mol) 4-nitrophthalic acid, slowly add 40.9mL (0.2613mol) isooctyl alcohol subsequently, add the dense H of 1.68mL while stirring at the bottom of bottle
2sO
4(0.0309mol) catalysis, 130 DEG C of stirring and refluxing, reaction mixture drips to tlc silica gel plate (2.5cm × 8.0cm) so that whether inspection reaction completes; (developer: dissolve a small amount of potassium permanganate in acetone solvent till raw material point disappears is detected until thin plate chromatography (TLC); Developping agent, normal hexane: ethyl acetate=5:1); React after stirring and refluxing 6h;
The water of the distillation unreacted isooctyl alcohol of removing and generation; Now liquid is poured in frozen water while hot, leave standstill for some time layering after magnetic agitation 5min, by lower floor yellow oily liquid 10%Na
2cO
3solution washing to water layer is colourless (pH7.0-8.0); Finally, crude oil is dry with anhydrous sodium sulfate dehydration, obtains yellow oily liquid 4-nitrophthalic acid two (2-ethyl) own ester and DEHP haptens intermediate.4-nitrophthalic acid two (2-ethyl) own ester molecule formula: C
24h
37nO
6, molecular weight: 435.55; Productive rate: 92.8%; Purity 97%.
the sign of embodiment 4, phthalic acid two (2-ethyl) own ester hapten intermediate
DEHP haptens intermediate 4-nitrophthalic acid two (2-ethyl) the own ester obtained, its structure is by the detection means such as infrared spectra, UV spectrum Analysis and Identification, as shown in Figure 1, its feature is as follows for the infrared spectrogram of DEHP haptens intermediate: IR (KBr) ν/cm
-1: 1532.84,1350.78 (-NO
2), 1731.20 (C=O), 1278.95,1128.22 (C-O-C), 2928.61,2860.47,2732.86 (-CH
3), 1611.98,1463.37,1350.78 (C=C), 1412.61 (d-OCH
2cH (CH
2cH
3) CH
2cH
2cH
2-), 3048.89,854.03 (C-H, Ar).Analytical results is known ,-the NO of 4-nitrophthalic acid two (2-ethyl) own ester
2asymmetrical stretching vibration (ν
as-
nO2) absorption band appear at 1532.84cm
-1near, symmetrical stretching vibration (ν
s-
nO2) absorption band appear at 1350.78cm
-1near; In addition, aryl esters ν
c=oabsorption peak obviously appears at 1731.20cm
-1place, the biobelt of the stretching vibration of phthalic ester appears at 1278.95,1128.22cm
-1place; The ν of virtue core
c=cabsorption band appears at 1611.98,1463.37,1350.78cm
-1place.All in all, above-mentioned crest has absolutely proved in reaction product the constitutional features possessing phenyl ring, (2-ethyl) hexyloxy, nitro functions.
the Preparation and characterization of embodiment 5, phthalic acid two (2-ethyl) own ester hapten
It is bottom 500mL tri-mouthfuls of round-bottomed flasks that 2.0g (0.0046mmol) DEHP haptens intermediate (4-nitrophthalic acid two (2-ethyl) own ester) is encased in volume, 230mL (2.5871mol) benzene is added while stirring subsequently at the bottom of bottle, to be mixed evenly after add 2.8g (0.0428mol) pure zinc powder again, after stirring, gradation adds the dense HCl of 8.2mL (0.0984mol) again, 2.8g (0.0428mol) pure zinc powder is again added, 25 DEG C of stirring reaction 11h after 25 DEG C of stirring 15min;
After reaction terminates, 230mL cold water is added reaction system, and is neutralized to weakly alkaline (pH7.0-8.5) with 1M sodium hydroxide solution; Isolate benzene layer after leaving standstill 1h, then use benzene aqueous layer extracted, combining extraction liquid, after washing, use anhydrous Na
2sO
4dehydrate; Extraction liquid underpressure distillation is to remove benzene, through silica gel column chromatography, (eluent is the yellow solid obtained, the mixed solution (V/V=15:1) of acetic acid and normal hexane) underpressure distillation afterwards, obtain yellow crystals 4-aminophthalic acid two (2-ethyl) own ester, i.e. DEHP haptens.Phthalic acid two (2-ethyl) own ester hapten molecular formula: C
24h
39nO
4, molecular weight: 405.57; Productive rate 65.2%; Fusing point: 34 ~ 36 DEG C; Purity 98%.
the preparation of embodiment 6, phthalic acid two (2-ethyl) own ester hapten
It is bottom 500mL tri-mouthfuls of round-bottomed flasks that 2.0g (0.0046mmol) DEHP haptens intermediate (4-nitrophthalic acid two (2-ethyl) own ester) is loaded volume, subsequently to adding 227mL (2.5534mol) benzene at the bottom of bottle while stirring, 3.0g (0.0460mol) pure zinc powder is added again after haptens is dissolved by benzene, after stirring, gradation adds the dense HCl of 7.7mL (0.0924mol), 3.0g (0.0460mol) pure zinc powder is again added, 28 DEG C of stirring reaction 12h after stirring at room temperature 15min;
After reaction terminates, 227mL cold water is added reaction system, and is neutralized to weakly alkaline (pH7.0-8.5) with 1M sodium hydroxide solution; Isolate benzene layer after leaving standstill 1h, then use benzene aqueous layer extracted, combining extraction liquid, after washing, use anhydrous Na
2sO
4dehydrate, extraction liquid underpressure distillation is to remove benzene, through silica gel column chromatography, (eluent is the yellow solid obtained, the mixed solution (V/V=15:1) of acetic acid and normal hexane) underpressure distillation afterwards, obtain yellow crystals 4-aminophthalic acid two (2-ethyl) own ester, i.e. DEHP haptens.Phthalic acid two (2-ethyl) own ester hapten molecular formula: C
24h
39nO
4, molecular weight: 405.57; Productive rate 66.1%; Fusing point: 34 ~ 36 DEG C; Purity 98%.
the preparation of embodiment 7, phthalic acid two (2-ethyl) own ester hapten
It is bottom 500mL tri-mouthfuls of round-bottomed flasks that 2.0g (0.0046mmol) DEHP haptens intermediate (4-nitrophthalic acid two (2-ethyl) own ester) is loaded volume, subsequently to adding 239mL (2.6884mol) benzene at the bottom of bottle while stirring, 3.3g (0.0511mol) pure zinc powder is added again after haptens is dissolved by benzene, after stirring, gradation adds the dense HCl of 8.6mL (0.1032mol), 3.3g (0.0511mol) pure zinc powder is again added, 30 DEG C of stirring reaction 10h after stirring at room temperature 15min;
After reaction terminates, 239mL cold water is added reaction system, and is neutralized to weakly alkaline (pH7.0-8.5) with 1M sodium hydroxide solution; Isolate benzene layer after leaving standstill 1h, then use benzene aqueous layer extracted, combining extraction liquid, after washing, use anhydrous Na
2sO
4dehydrate, extraction liquid underpressure distillation is to remove benzene, through silica gel column chromatography, (eluent is the yellow solid obtained, the mixed solution (V/V=15:1) of acetic acid and normal hexane) underpressure distillation afterwards, obtain yellow crystals 4-aminophthalic acid two (2-ethyl) own ester, i.e. DEHP haptens.Phthalic acid two (2-ethyl) own ester hapten molecular formula: C
24h
39nO
4, molecular weight: 405.57; Productive rate 67.3%; Fusing point: 34 ~ 36 DEG C; Purity 98%.
the sign of embodiment 8, phthalic acid two (2-ethyl) own ester hapten
DEHP haptens 4-aminophthalic acid two (2-ethyl) the own ester obtained, its structure is by detection means Analysis and Identification such as nucleus magnetic resonance, infrared spectra, UV spectrum, as shown in Figure 2, its feature is as follows for the haptenic infrared spectrogram of DEHP: IR (KBr) ν/cm
-1: 3473.78,3376.37 (-NH
2), 1714.16 (C=O), 1280.38,1127.72 (C-O-C), 2958.83,2930.61,2873.27,2860.21 (-CH
3), 1463.19 (d-OCH
2cH (CH
2cH
3) CH
2cH
2cH
2-), 1603.78,1569.83,1382.04 (C=C), 1628.00,835.80 (C-H, Ar).Analytical results is known, the asymmetrical stretching vibration (ν of the N-H key of the primary amine of 4-aminophthalic acid two (2-ethyl) own ester
asN-H) and symmetrical stretching vibration (ν
sN-H) absorption band appear at 3473.78 and 3376.37cm respectively
-1near, this has confirmed that nitro is reduced into amino; In addition, aryl esters ν
c=oabsorption peak obviously appears at 1714.16cm
-1place, the biobelt of the stretching vibration of phthalic ester appears at 1280.38,1127.72cm respectively
-1place; The ν of virtue core
c=cabsorption band appears at 1603.78,1569.83,1382.04cm
-1place.All in all, above-mentioned crest has absolutely proved in reaction product the constitutional features possessing phenyl ring, (2-ethyl) hexyloxy, amido functional group.
The data results of its nucleus magnetic hydrogen spectrum (see Fig. 3) is as follows:
1hNMR (400MHz, CDCl
3): δ 7.68 (d, 1H, ArH), 7.24 (d, 1H, ArH), 6.76 (d, 1H, ArH), 4.19 (b, 2H ,-NH
2), 4.14 (t, 2H, OCH
2), 4.12 (t, 2H, OCH
2), 1.64 (m, 2H, OCH
2cH), 1.62 – 1.04 (m, 16H, OCH
2cH (CH
2cH
3) CH
2cH
2cH
2), 0.95 – 0.87 (t, 12H, OCH
2cH
2(CH
2cH
3) CH
2cH
2cH
2cH
3) ppm.Therefrom can obviously find out: after phenyl ring being introduced amino coupled arm, there is amino hydrogen in δ 4.19 place, and the position of the hydrogen atom occurred in wave spectrogram conforms to the structure of number with synthesized haptens 4-aminophthalic acid two (2-ethyl) own ester.
On the whole, the result of infrared spectra and proton nmr spectra qualification is all presented in Benzene Molecule structure and successfully introduces amino, by itself and protein generation coupling and then can synthesize the artificial holoantigen of DEHP.
the preparation of embodiment 9, the artificial holoantigen of phthalic acid two (2-ethyl) own ester
Phthalic acid two (2-ethyl) own ester hapten prepared by the present invention, is mainly used in the immunodetection of phthalic acid two (2-ethyl) own 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 phthalic acid two (2-ethyl) own ester on this basis.Below with regard to phthalic acid two (2-ethyl) own ester hapten make be used as following applicating example:
Adopt diazotization legal system for DEHP immunogen DEHP-BSA, concrete synthesis step: 0.0406g (0.1mM) 4-aminophthalic acid two (2-ethyl) own ester 100 μ LDMF are dissolved, then dropwise joining in the 25mL Erlenmeyer flask of the mixed solution that 40 μ L concentrated hydrochloric acids and 900 μ LDMF are housed when constantly stirring, cooling in the rearmounted ice bath of thing heating for dissolving to be mixed; Now, when 4 DEG C of low temperature stir, dropwise drip 1M sodium nitrite solution, controlling such as reaction acidity with pH test paper is 2.0-3.0, simultaneously with starch potassium iodide paper colour developing, developing time is the 1-3s after dripping, and stops dripping when test paper becomes dusty blue instantaneously by white, continue reaction 60min again, add 1.0g urea to remove unreacted Sodium Nitrite; Then, when 4 DEG C of low temperature stir, above-mentioned diazonium salt is dropwise joined in 0.08mMBSA solution (dissolving of 0.01MpH9.18 sodium borate buffer liquid) 10mL, solution 1M sodium hydroxide solution regulates pH until gradually in orange red, after continuing reaction 12h, the antigen crude product that obtains is loaded dialysis tubing, be placed in 0.01MpH7.40 phosphate buffered saline buffer and dialyse 3d, water is changed 1 time, finally by end product 4000rmin every 8h
-1off-line 10min, gets its supernatant liquor and DEHP immunogen DEHP-BSA.Immunogen after ultraviolet-visible spectrophotometer qualification, in a small amount packing ,-20 DEG C of lyophilizes, packing in-20 DEG C of preservations.Lyophilize, packing in-20 DEG C of preservations.
By glutaraldehyde legal system DEHP for coating antigen DEHP-OVA, concrete synthesis step: it is in the Erlenmeyer flask of 25mL that own for 0.0406g (0.1mM) 4-aminophthalic acid two (2-ethyl) ester is loaded volume, limit drips 1mLN, dinethylformamide (DMF) limit is stirred, dropwise join in 0.08mMOVA solution (dissolving of 0.01MpH7.40 phosphate buffered saline buffer) 10mL after stirring again, 0.038mL25% glutaraldehyde is slowly added subsequently in this mixed system, after 4 DEG C of low temperature black out stirring reaction 24h, the antigen crude product obtained is loaded dialysis tubing, be placed in 0.01MpH7.40 phosphate buffered saline buffer low temperature 4 DEG C dialysis 3d, water is changed 1 time every 8h, finally by end product 4000rmin
-1centrifugal 10min, gets its supernatant liquor and DEHP coating antigen DEHP-OVA.Coating antigen after ultraviolet-visible spectrophotometer qualification, lyophilize, in a small amount packing ,-20 DEG C of freezen protective.
Conjugate is after ultraviolet-visible pectrophotometer scanning qualification, as can be seen from the DEHP haptens of Fig. 4 and Fig. 5, carrier proteins and artificial holoantigen ultraviolet spectrogram, DEHP haptens has two ultraviolet absorption peaks and 286nm and 309nm, and wherein 309nm place ultraviolet absorption peak is characteristic peak; The charateristic avsorption band of carrier proteins BSA is positioned at 227nm and 278nm place, and wherein the absorption value at 278nm ultraviolet absorption peak place is very low; Carrier proteins OVA has two ultraviolet absorption peaks, lays respectively at 234nm and 279nm place, and wherein the absorption value at 279nm ultraviolet absorption peak place is lower.Conjugate DEHP-BSA has two ultraviolet absorption peaks, lays respectively at 237nm and 329nm place; Conjugate DEHP-OVA has two ultraviolet absorption peaks, lays respectively at 229nm and 343nm place.DEHP-BSA and DEHP-OVA haptens charateristic avsorption band there occurs red shift, and this may be caused by the impact of the absorption peak being subject to carrier proteins.The appearance of absorption peak Red Shift Phenomena, above information shows DEHP-BSA and DEHP-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.
the preparation of embodiment 10, phthalic acid two (2-ethyl) own ester polyclonal antibody
Select 2 adult healthy male New Zealand rabbits, the artificial immunogen DEHP-BSA synthesized is after fully mixing with Freund's complete adjuvant, neck and back point-like injecting immune are carried out to White Rabbit, after 7 booster immunizations, adopt indirect enzyme-linked immunosorbent assay antiserum titre, its antiserum titre reaches 150000 all.Test display cross reaction is all not obvious, illustrates that its specificity is better.
phthalic acid two (2-in embodiment 11, indirect competition BA-ELISA immune analysis method testing environment sample
ethyl) own ester
Concrete steps are as follows: with being buffered liquid (0.05MpH9.60 carbonate buffer solution) suitably dilution coating antigen solution (DEHP-OVA) with bag, 100 μ L/ holes, add in 96 hole enzyme plates, and 4 DEG C of bags are spent the night; Next day outwells coating buffer, and every hole adds 200 μ L washingss (0.01MpH7.40PBST), repeated washing three times, each 3min; After patting dry enzyme plate with thieving paper, every hole adds confining liquid 200 μ L, 37 DEG C of incubations 1 hour; Outwell confining liquid, repeat above-mentioned washing process three times, the anti-DEHP polyclonal antibody (Bio-pAb-DEHP) of biotinylation is diluted to proper concn, every hole adds 50 μ L biotinylated antibodies and 50 μ LDEHP standard models (PBS (v/v) gradient dilution with containing 5% methyl-sulphoxide (DMSO)) successively, the every hole of blank well adds 100 μ LPBS damping fluids, 37 DEG C of incubations 30 minutes; Outwell antigen-antibody reaction liquid, repeated washing three times, suitably dilute with PBST the Streptavidin (HRP-SA) that horseradish peroxidase (HRP) marks, every hole adds 100 μ L, 37 DEG C of incubations 1 hour; Outwell HRP-SA solution, repeated washing five times, every hole adds freshly prepared nitrite ion (400 μ L2.5mg/mL3,3 ', 5, the H of 5 '-tetramethyl benzidine substrate solution and 10mL phosphoric acid salt-citric acid substrate buffer solution, 10 μ L30%
2o
2be mixed to form nitrite ion) 100 μ L/ holes, lucifuge reaction 15min under room temperature; Every hole adds 50 μ L stop buffers (2mol/L sulphuric acid soln) with color development stopping, then measures each hole in 450nm and 630nm place absorbance (OD value), with OD=OD by microplate reader
450-OD
630as final reading.Experimental result inhibiting rate represents, and is ordinate zou with inhibiting rate, and the logarithmic value of standard model concentration is X-coordinate drawing standard curve, and the standard working curve of foundation as shown in Figure 6.Wherein, inhibiting rate (%)=(1-A/A
0) × 100, wherein A is the OD value in the hole having sample to exist, A
0for there is no the OD value in the hole of sample.
Being prepared as follows of biotinylated antibody: with 0.05MpH9.60 carbonate buffer solution, the anti-DEHP polyclonal antibody after purifying is diluted to about 1.0mg/mL, gets 2mL and add in Erlenmeyer flask; Prepare 1.0mg/mL vitamin H-N-succinimido ester (BNHS) solution with DMSO, in above-mentioned Erlenmeyer flask, add BNHS solution, make the mass ratio of BNHS:IgG be 1:10, stirred at ambient temperature reacts 4 hours; After reaction terminates, reaction solution is loaded in dialysis tubing, with PBS damping fluid dialysis 3d, change water every day 3 times.Centrifugation obtains corresponding biotinylated antibody solution (Bio-pAb-DEHP), after the packing of antibody-solutions small volume, in-20 DEG C of freezen protective after removing a small amount of precipitation.
The typical curve equation that indirect competition BA-ELISA immune analysis method detects phthalic acid two (2-ethyl) own ester is Y=21.57LgC
dEHP+ 56.01, coefficient R=0.9832, wherein IC
50represent the sensitivity of detection method, analyte concentration 0.526 μ g/L corresponding when namely inhibiting rate is 50%; IC
10the Monitoring lower-cut of method for expressing, analyte concentration 0.0074 μ g/L corresponding when namely inhibiting rate is 10%; IC
20-IC
80represent linearity range, i.e. 0.021 μ g/L ~ 12.948 μ g/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. phthalic acid two (2-ethyl) own ester hapten, is characterized in that, structural formula is such as formula shown in (I):
2. a preparation method for the own ester hapten of phthalic acid two (2-ethyl) according to claim 1, described preparation method comprises the steps:
Haptens Intermediate Preparation: in strong acid environment, 4-nitrophthalic acid and isooctyl alcohol generation esterification, generate 4-nitrophthalic acid two (2-ethyl) own ester, be haptens intermediate;
Prepared by haptens: in non-proton organic solvent environment, described haptens intermediate and zinc powder, strong acid react, and obtain phthalic acid two (2-ethyl) own ester hapten.
3. the preparation method of the own ester hapten of phthalic acid two (2-ethyl) according to claim 2, it is characterized in that, the step of described haptens Intermediate Preparation is specially: be dissolved in by 4-nitrophthalic acid in isooctyl alcohol solution, be added dropwise to strong acid, the reaction system generation esterification of formation.
4. the preparation method of phthalic acid two (2-ethyl) the own ester hapten according to Claims 2 or 3, it is characterized in that, the mol ratio of 4-nitrophthalic acid, isooctyl alcohol, strong acid is 1:(5 ~ 6): (0.6 ~ 0.7); The temperature of described reaction is 120 ~ 150 DEG C, and the time of reaction is 5 ~ 6 hours; Described strong acid is the vitriol oil or concentrated hydrochloric acid.
5. the preparation method of phthalic acid two (2-ethyl) the own ester hapten according to Claims 2 or 3, it is characterized in that, the step of described haptens Intermediate Preparation also comprises:
A1, remove the water that isooctyl alcohol that esterification does not occur in described reaction system and reaction generate, then remainder poured in frozen water, precipitate, obtain haptens crude intermediate;
A2, be colourless by the washing of described haptens crude intermediate to washings, dry, obtain the haptens intermediate of purifying.
6. the preparation method of the own ester hapten of phthalic acid two (2-ethyl) according to claim 2, it is characterized in that, step prepared by described haptens is specially: be dissolved in by haptens intermediate in non-proton organic solvent environment; Then add zinc powder, mix; Be added dropwise to strong acid again, again add zinc powder, the reaction system isothermal reaction of formation.
7. the preparation method of phthalic acid two (2-ethyl) the own ester hapten according to claim 2 or 6, it is characterized in that, the mol ratio of described haptens intermediate, strong acid, nonionic organic solvent and zinc powder is 1:(20 ~ 25): (550 ~ 580): (18 ~ 20); The temperature of described isothermal reaction is 25 ~ 30 DEG C, and the time of described isothermal reaction is 10 ~ 12 hours; Described nonionic organic solvent is selected from benzene, toluene or acetone.
8. the preparation method of phthalic acid two (2-ethyl) the own ester hapten according to claim 2 or 6, it is characterized in that, described haptens is prepared further comprising the steps of:
After B1, described reaction terminate, cool described reaction system, adjustment pH is weakly alkaline;
B2, leave standstill, extract to obtain extraction liquid, wash described extraction liquid, dry;
Organic solvent constituent in B3, the dry rear extraction liquid of removing, obtains yellow solid;
B4, by gained yellow solid through silica gel column chromatography, then underpressure distillation, obtains phthalic acid two (2-ethyl) own ester hapten.
9. a purposes for the own ester hapten of phthalic acid two (2-ethyl) according to claim 1, is characterized in that, specifically for the detection of trace fluidizer phthalic acid two (2-ethyl) own ester.
10. one kind requires the holoantigen of phthalic acid two (2-ethyl) the own ester hapten described in 1 based on claim, it is characterized in that, described holoantigen is prepared by the following method: by phthalic acid two (2-ethyl) own ester hapten by glutaraldehyde method or diazotization method or carbodlimide method and carrier protein couplet, to obtain final product.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012046640A (en) * | 2010-08-27 | 2012-03-08 | Canon Inc | Pigment compound, pigment dispersant containing the same, pigment composition, pigment dispersion and toner |
| CN103097341A (en) * | 2010-08-27 | 2013-05-08 | 佳能株式会社 | Azo compound, pigment dispersant containing the azo compound, pigment composition, pigment dispersion, and toner |
| CN103760337A (en) * | 2013-12-11 | 2014-04-30 | 安徽师范大学 | Indirect competive enzyme-linked immunosorbent assay detection method of bis(2-ethylhexyl)phthalate |
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2015
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012046640A (en) * | 2010-08-27 | 2012-03-08 | Canon Inc | Pigment compound, pigment dispersant containing the same, pigment composition, pigment dispersion and toner |
| CN103097341A (en) * | 2010-08-27 | 2013-05-08 | 佳能株式会社 | Azo compound, pigment dispersant containing the azo compound, pigment composition, pigment dispersion, and toner |
| CN103760337A (en) * | 2013-12-11 | 2014-04-30 | 安徽师范大学 | Indirect competive enzyme-linked immunosorbent assay detection method of bis(2-ethylhexyl)phthalate |
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| Title |
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| RUIYAN SUN ETAL: "A sensitive heterogeneous biotin–streptavidinenzyme-linked immunosorbent assay for the determination of di-(2-ethylhexyl)phthalate (DEHP) in beverages using a specific polyclonal antibody", 《ANAL. METHODS》 * |
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