CN1528511A - Endotoxin adsorbent and preparation method thereof - Google Patents
Endotoxin adsorbent and preparation method thereof Download PDFInfo
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Abstract
本发明涉及一种内毒素吸附剂及其制备方法。本发明是以天然高分子或合成高分子为载体，以二甲基胺作配体，并在二甲基胺的β位连有羟基，吸附剂的胺基含量为10－130μmol/g吸附剂。由于这种结构与内毒素之间可同时形成离子键、氢键、和八元环，且内毒素和吸附剂的疏水链处在八元环的同侧，因此对内毒素有较强的识别能力和吸附能力。吸附剂可用于生化制品中内毒素的去除，也可用于血液灌流去除患者血液中的内毒素，属于新一代产品，使用本发明毒副作用小，成本低，价格便宜，适合在国内推广。The invention relates to an endotoxin adsorbent and a preparation method thereof. The present invention uses natural macromolecule or synthetic macromolecule as the carrier, dimethylamine as the ligand, and a hydroxyl group connected to the β position of the dimethylamine, and the amine group content of the adsorbent is 10-130 μmol/g adsorbent . Since this structure can form ionic bonds, hydrogen bonds, and eight-membered rings with endotoxin at the same time, and the hydrophobic chains of endotoxin and adsorbent are on the same side of the eight-membered ring, it has a strong recognition of endotoxins capacity and adsorption capacity. The adsorbent can be used for the removal of endotoxin in biochemical products, and can also be used for hemoperfusion to remove endotoxin in patient's blood. It belongs to a new generation of products. The use of the present invention has small toxic and side effects, low cost and low price, and is suitable for domestic promotion.
Description
Technical field
The present invention relates to biochemical the separation and biomedical materials field, particularly a kind of endotoxin absorbent and preparation method thereof.
Background technology
Endotoxemia is to cause owing to the endotoxin on the outer cell membrane of Gram-negative bacteria is discharged in the blood, can come across [Wang Jinda in the multiple lysis, China's critical illness emergency medicine, 1998,10 (10): 578], as: diseases such as large-area burns, serious hepatitis, cirrhosis, body's immunity is badly damaged, and can cause a series of serious pathological changes such as shock, disseminated intravascular coagulation, MOF, finally cause organ necrosis, irreversible shock and death.The U.S. all have every year surpass 100,000 people die from this disease (Skelter et al, Arch.Microbiol.1995,164,383-389.), still do not have effective methods of treatment clinically.How accomplishing to remove in time, effectively or destroy the interior endotoxin of patient's body, is the key issue of treatment endotoxemia.Developed molecular biosciences preparation (the Lopukhin M A of multiple anti-endotoxin in recent years abroad, Am JGastroenterol, 1997,68:345), but because the complex manufacturing of antibody, the medicine source is few, cost is high, price is very expensive, and still do not have up to now a kind of can pass through III phase clinical trial (Yao Yongming etc. Chinese critical illness emergency medicine, 1999,11:456).
To some great difficult venereal disease diseases, the blood purification therapy has unique curative effect, be the indispensable important means of clinical treatment, wherein the blood perfusion therapy is to rely on adsorbent directly to adsorb toxic substance or the morbid substance of removing in the blood, reaches the purpose that purifies the blood, alleviates and treat disease.The method treatment endotoxemia that domestic and international researcher has begun one's study and utilized blood purification, people such as Cheong Kuoc Va [Chinese critical illness emergency medicine, 1997,9 (4): 193] once use active carbon that the animal of endotoxin modeling is carried out whole blood perfusion, found to have certain effect.But,, inevitably can adsorb a large amount of compositions useful, and clearance rate is lower to human body in the endotoxic while of absorption because active carbon is a kind of wide spectrum adsorbent.The researcher of Japan is coated on PB on the inwall of blood filter doughnut, can remove the endotoxin [AtificialOrgans 22 (12) for Tani T, et al.: 1038-1044 (1998)] in the blood plasma preferably.But PB costs an arm and a leg and has a renal toxicity, if from the carrier toxic side effect that comes off, and said method need carry out plasma perfusion with blood plasma with after blood cell separates, and expense is very expensive, is not suitable for promoting at home.
Biological products very easily are subjected to endotoxic pollution in process of production, thereby the endotoxin of removing in the biological products is the biomedicine field a great problem.In recent years, Chinese scholars is removed in biological products and has been obtained some achievements aspect the endotoxin.Method commonly used has ultrafiltration, two-phase extraction method, absorption method etc., and wherein the absorption of the affinity in the absorption method is the endotoxic a kind of very promising method of selective absorption, selects specific absorption part for use, the selective absorption endotoxin.The affinity ligand of having reported has PB, histidine, polycation part, deoxycholic acid etc., and part mainly combines with endotoxin by ionic bond and hydrophobic effect, reaches to remove endotoxic purpose.
Summary of the invention
The purpose of this invention is to provide a kind of novel endotoxin absorbent and preparation method thereof, this endotoxin absorbent has used the Novel Ligands structure, endotoxin there are higher recognition capability and adsorption capacity, be fixed on natural polymer or the synthetic high polymer carrier, can be used for endotoxic removing in biochemical product and the blood.
The present invention is to be carrier with spherical natural polymer or synthetic high polymer, and by the adsorbent that the arm linking ligand constitutes, the amido content of adsorbent is 10-130 μ mol/g adsorbent.
Described part is a dimethyl amine; Described arm is a saturated aliphatic chain; Described ball type carrier is shitosan, agar, cellulose or polymethacrylates.
The β position of described dimethyl amine is connected with hydroxyl.
Described ball type carrier is a polymethyl methacrylate.
Described saturated aliphatic chain is a diamines.Described diamines is ethylenediamine, propane diamine, butanediamine, pentanediamine or hexamethylene diamine.
With spherical polymethyl methacrylate is that the preparation method of the endotoxin absorbent of carrier is through following step:
(1) the spherical polymethyl methacrylate of carrier is synthetic
In methyl methacrylate that contains 0.1～0.5g benzoyl peroxide (initator) and divinylbenzene mixed liquor, join the aqueous phase that contains 10～25% common salts and 0.1～0.6% gelatin, add several 0.05～0.2% methine orchids, stir, be warming up to 50～70 ℃, polymerization 1～3 hour; Be warming up to 72～78 ℃ again, polymerization 1～3 hour; Be warming up to 82～89 ℃ again, polymerization 1～3 hour; Be warming up to 92～98 ℃ again, constant temperature 4～6 hours.
(2) introduce arm
Make solvent with DMF, plexiglass and excessive diamine reactant reacted 7～10 hours down at 100～150 ℃.
(3) epoxychloropropane activation
Add excessive epoxychloropropane and sodium hydroxide solution in the above-mentioned product, swelling is spent the night, and reacts 4～6 hours down at 25～60 ℃, makes the epoxychloropropane activation products.
(4) introduce primary amine groups
Epoxychloropropane activation products and excessive ammonia or diamine reactant reacted 7～10 hours down at 100～150 ℃, and the product that obtains connects primary amine groups at the epoxychloropropane end.
(5) primary amino radical is changed into tertiary amine groups
Add excessive formic acid and formaldehyde, be heated to boiling, do not emerge to there being bubble, through washing, ethanol washing, drying obtains the adsorbent that end group is a dimethyl amine.
The material proportion of above-mentioned each step of preparation method is: methyl methacrylate: divinylbenzene (W: W) be 3～5: 7 (g: g); Plexiglass: diamines (W: V) be 1: 5～15 (g: ml); Introduce the carrier of arm: epoxychloropropane (W: V) be: 1: 5～20 (g: ml); Epoxy activated carrier: ammonia or diamines (W: V) be 1: 5～15 (g: ml); Concentration of sodium hydroxide solution is 0.55mol/L, 2～5ml; The carrier of primary amine groups: formic acid: formaldehyde (W: V: V) be: 1: 5～15: 5～15 (g: ml: ml).
The spherical shell glycan is that the preparation method of the endotoxin absorbent of carrier is through following step:
(1) spherical chitin carrier is synthetic
The shitosan aqueous acetic acid, adding is dissolved with in the toluene solution of 0.5～2.5g Span 80, stirs 0.5～2.5 hour.Slowly drip 20～60ml glutaraldehyde water solution, be warming up to 35～55 ℃, constant temperature 0.5～1.5 hour.Add sodium hydrate aqueous solution, the pH of conditioned reaction system is an alkalescent.Be warming up to 70～85 ℃, constant temperature 3～5 hours.Use the absolute ethyl alcohol extract product, washing is weighed, and the carrier of different sizes is collected in screening.Use NaBH under the room temperature alkali condition
4Reduction 12～36h cleans standby.
(2) epoxychloropropane activated carrier
Add excessive epoxychloropropane, methyl-sulfoxide and sodium hydroxide solution in the above-mentioned product, swelling is spent the night, and reacts 4～6 hours down for 60 ℃ at 251～5% shitosan aqueous acetic acids, makes the epoxychloropropane activation products.
Each step synthetic method is with polymethyl methacrylate carrier (3), (4) step later on.
The material proportion of above-mentioned each step of preparation method is: shitosan: acetic acid: water (W: V: be 1～5 V): 3: 100 (g: ml: ml); Chitin carrier: epoxychloropropane: methyl-sulfoxide (W: V: V) be 1: 1～5: 5～10 (g: ml: ml), NaOH 2M, 1～5ml; Epoxy activated carrier: ammonia or diamines (W: V) be 1: 5～15 (g: ml); The carrier of primary amine groups: formic acid: formaldehyde (W: V: V) be: 1: 5～15: 5～15 (g: ml: ml).
The preparation method of the endotoxin absorbent of spherical agar carrier is through following step
(1) the spherical agar carrier of preparation:
Aqueous agar solution heat fused with 2～10%, pour in toluene-carbon tetrachloride system, add Tween 80, stir, make agar solution in organic facies, be dispersed into drop, 60～90 ℃ were reacted 2～4 hours, after the cooling, topple over and the upper strata organic solvent, the agar ball of screening 0.45～1.0mm, after water fully washs, as adsorbing agent carrier.
(2) epoxychloropropane activated carrier:
Carrier in 2M NaOH solution, 30～50 ℃ down with epichlorohydrin reaction 1～4 hour, wash with water to neutrality, remove unreacted epoxychloropropane, obtain the carrier of epoxychloropropane modification.
Each step synthetic method is with polymethyl methacrylate carrier (3), (4) step later on.
The material proportion of each step of preparation method of above-mentioned endotoxin absorbent is: toluene: carbon tetrachloride (V: V) be 1～4: 1 (ml: ml); The NaOH of agar ball: 2M: epoxychloropropane (W: V: V) be 1: 1～4: 0.5～2 (g: ml: ml); Epoxy activated carrier: ammonia or diamines (W: V) be 1: 5～15 (g: ml); The carrier of primary amine groups: formic acid: formaldehyde (W: V: V) be: 1: 5～15: 5～15 (g: ml: ml).
The preparation method of the endotoxin absorbent of ball shaped cellulose carrier is through following step
(1) the plain carrier of dimension is synthetic
Cotton linter is at first drifted with 1% aqueous sodium hypochlorite solution, puts into 19% sodium hydroxide solution again, and soaking at room temperature 2 hours is extracted, sealing was worn out three days under the room temperature, got sample and measured dry weight, with the carbon disulfide that measures, 25 ℃ were reacted 5 hours down, obtain orange red thick liquid then; With the chlorobenzene is decentralized medium, regulate decentralized medium proportion with carbon tetrachloride, potassium oleate is a dispersant, add calcium carbonate powder in the viscose as pore-foaming agent, adopt suspension polymerization, 80～95 ℃ were reacted 1～3 hour, washing, remove pore-foaming agent with the saturated calcium chloride solution of 0.5N hydrochloric acid (including 20% sodium chloride) hydrolysis, be washed to neutrality, the screening different meshes is standby.
Each step synthetic method is with agar carrier (2), (3) and (4) step later on.
The material proportion of each step of preparation method of above-mentioned endotoxin absorbent is: 75～100% (W: W) chlorobenzene, 0～25% (W: W) carbon tetrachloride, 0.2% (W: W) potassium oleate, form organic decentralized photo; Take by weighing the viscose of organic facies 1/3 weight, (W: calcium carbonate W) (60 μ m) powder is as pore-foaming agent to wherein adding 0～10%; The NaOH of cellulose balls: 2M: epoxychloropropane (W: V: V) be 1: 1～4: 0.5～2 (g: ml: ml); Epoxy activated carrier: ammonia or diamines (W: V) be 1: 5～15 (g: ml); The carrier of primary amine groups: formic acid: formaldehyde (W: V: V) be: 1: 5～15: 5～15 (g: ml: ml).
Described endotoxin absorbent is removed endotoxic method from biological products: under the room temperature, adsorbent is contained in the endotoxic biological products solution direct the adding, vibration absorption 15 minutes, adsorbent: solution (W: V) be 1: 20 (g: ml), or adsorbent: serum (W: V) be 1: 4 (g: ml).Detect absorption front and back endotoxin concns with dynamic method.
The method that described endotoxin absorbent carries out blood perfusion is: under the room temperature, get adsorbent and pack in the perfusion post, endotoxemia patient's whole blood is with the flow velocity perfusion of 15ml/min 2 hours, adopts azo development process to detect endotoxin concns before and after the absorption; Or under the room temperature, adsorbent is contained in the endotoxic blood plasma static absorption 20 minutes direct the adding.
The endotoxin absorbent that the present invention is prepared has the spaced structure of polarity and non-polar group on its spacerarm.Little heterogeneous surperficial hypothesis (Microheterogeneous surface concept) thinks, the block copolymer with hydrophilic/hydrophobic domain structure can significantly suppress hematoblastic and stick and activate, and presents anticoagulation function preferably in the experiment in vivo.Many artificial synthetic triblock copolymers confirm that this class material has good anticoagulation function.On the spacerarm of the endotoxin absorbent that we synthesized, also has similar hydrophilic/hydrophobic domain structure.The blood compatibility that this helps improving adsorbent, making it can clinical practice.
The present invention is to be carrier with natural polymer or synthetic high polymer, makes part with dimethyl amine, is connected with hydroxyl in the β position of dimethyl amine.Owing to can form ionic bond, hydrogen bond and octatomic ring simultaneously between this structure and the endotoxin, and the hydrophobic chain of endotoxin and adsorbent is in the homonymy of octatomic ring, therefore endotoxin had stronger recognition capability and adsorption capacity.This ligand structure, with be both dimethyl amine and make part, the structure of no hydroxyl is compared on the β position, the former is than high nearly 10 times of the latter's adsorbance, the synergy of ionic bond, hydrogen bond, hydrophobic force and octatomic ring is described, than only more effective to endotoxic identification and suction-operated by ionic bond and hydrophobic force.Computer simulation results shows that the hydroxyl on the adsorbent only when the β position of part dimethyl amine, just has aforesaid synergy, sees the explanation of accompanying drawing subordinate list.Adsorbent can be used for endotoxic removal in the biochemical product, also can be used for the endotoxin in the hemoperfusion removal blood samples of patients.
In order to compare with traditional endotoxin absorbent, we are carrier with the polymethyl methacrylate, have synthesized identical arm, and part is respectively the adsorbent of histidine, lysine and alanine.Test through the water adsorption experiment, when part content fixedly the time, the adsorption effect that the β position is associated with the dimethyl amine part adsorbent of hydroxyl is is 4.7 times of part adsorbents adsorb amount with the histidine, be 4.2 times of part adsorbents adsorb amount with lysine, be to be 7.1 times of part adsorbents adsorb amount with the alanine.
The present invention is part with the dimethyl amine, and be associated with the structure of hydroxyl in its β position, used the Novel Ligands structure, endotoxin there are higher recognition capability and adsorption capacity, can this structure be incorporated on spherical agar, cellulose, shitosan or the polymethacrylates carrier by arm, form endotoxin absorbent, can be used for endotoxic removing in biochemical reagents or the blood.Use toxic and side effect of the present invention little, cost is low, and low price is adapted at domestic popularization.
Description of drawings
Fig. 1: adsorbent ligand structure schematic diagram.
When there is hydroxyl in the β position of Fig. 2 dimethyl amine, the synergy schematic diagram of computer simulation ionic bond, hydrogen bond, hydrophobic force and octatomic ring.
Fig. 3: the hydrogen bond when hydroxyl is in diverse location forms the situation schematic diagram.
The specific embodiment:
The endotoxin absorbent of the present invention's preparation in water and blood plasma, all has the good adsorption effect to endotoxin, can be illustrated by following embodiment, but not be that the present invention is imposed any restrictions.
Embodiment 1
The preparation of 1-1 polymethacrylates carrier
In the there-necked flask that agitator, reflux condensing tube, thermometer are housed, the methyl methacrylate 8.1g that will contain the 0.414g benzoyl peroxide, with divinylbenzene 18.8g mixed liquor, join in the water (volume is 2～4 times of organic facies) that contains 20% common salt and 0.5% gelatin, add 5～8 0.1% methine orchids.Regulate mixing speed, treat liquid pearl size to fit, epigranular is warming up to 67 ℃, polymerization two hours; Be warming up to 75 ℃ again, polymerization two hours; Be warming up to 85 ℃ again, polymerization two hours; Be warming up to 95 ℃ again, constant temperature four hours makes carrier PMMA.Wash by rubbing with the hands repeatedly through hot water, the absolute ethyl alcohol extracting is dry after 8 hours, and screening collection cut size size is that the carrier of 280～900 μ m is standby.
1-2 introduces arm
1-2-1 takes by weighing 1g PMMA, and 15ml DMF makes solvent, adds 10ml 1, the 2-ethylenediamine, and swelling is spent the night.Reacted 9 hours down at 130 ℃, make 1, the polymethyl methacrylate that 2-is ethylene diamine-modified.Through washing, ethanol washing, drying for standby.With determination of acid-basetitration primary amine groups content is 95.4 μ mol/g.
1-2-2 takes by weighing 1g PMMA, and 15ml DMF makes solvent, adds 10ml 1, the 3-propane diamine, and swelling is spent the night.Reacted 9 hours down at 130 ℃, make 1, the plexiglass that the 3-propane diamine is modified.Through washing, ethanol washing, drying for standby.With determination of acid-basetitration primary amine groups content is 94.7 μ mol/g.
1-2-3 takes by weighing 1g PMMA, and 15ml DMF makes solvent, adds 10ml 1, the 4-butanediamine, and swelling is spent the night.Reacted 9 hours down at 130 ℃, make 1, the plexiglass that the 4-butanediamine is modified.Through washing, ethanol washing, drying for standby.With determination of acid-basetitration primary amine groups content is 89.6 μ mol/g.
1-2-4 takes by weighing 1g PMMA, and 15ml DMF makes solvent, adds 10ml 1, the 5-pentanediamine, and swelling is spent the night.Reacted 9 hours down at 130 ℃, make 1, the plexiglass that the 5-pentanediamine is modified.Through washing, ethanol washing, drying for standby.With determination of acid-basetitration primary amine groups content is 90.5 μ mol/g.
1-2-5 takes by weighing 1g PMMA, and 15ml DMF makes solvent, adds 10ml 1, the 6-hexamethylene diamine, and swelling is spent the night.Reacted 9 hours down at 130 ℃, make 1, the plexiglass that the 6-hexamethylene diamine is modified.Through washing, ethanol washing, drying for standby.With determination of acid-basetitration primary amine groups content is 93.2 μ mol/g.
The activation of 1-3 epoxychloropropane
1-3-1 takes by weighing 1g1, and the plexiglass that 2-is ethylene diamine-modified adds the 2.5ml epoxychloropropane, and 4ml concentration is 0.55N NaOH, and 40 ℃ were reacted 6 hours, and made epoxy chloropropionate alkanisation 1, the plexiglass that 2-is ethylene diamine-modified.Through washing, ethanol washing, drying for standby.Get 1.0 grams and clean the gel ball of draining, add 3.0 milliliters of 1.3M hypo solutions, splash into 2 bromo bromothymol blue indicator (0.1% 20% ethanolic solution).Room temperature was placed after 30 minutes, under electromagnetic agitation with 0.01M standard salt acidometric titration to neutral, solution colour is terminal point by the blue stain Huang, and is contrast with 1.0 milliliters of non-activated gel carriers, calculate epoxy group content by the hydrochloric acid that consumes, getting the epoxy supported quantity is 88.2 μ mol/g.
1-3-2 takes by weighing 1g1, and the plexiglass that the 3-propane diamine is modified adds the 2.5ml epoxychloropropane, and 4ml concentration is 0.55N NaOH, and 40 ℃ were reacted 6 hours, made epoxy chloropropionate alkanisation 1, the plexiglass that the 3-propane diamine is modified.Through washing, ethanol washing, drying for standby.The same 1-3-1 of method of testing, recording the epoxy supported quantity is 87.3 μ mol/g.
1-3-3 takes by weighing 1g1, and the plexiglass that the 4-butanediamine is modified adds the 2.5ml epoxychloropropane, and 4ml concentration is 0.55N NaOH, and 40 ℃ were reacted 6 hours, made epoxy chloropropionate alkanisation 1, the plexiglass that the 4-butanediamine is modified.Through washing, ethanol washing, drying for standby.The same 1-3-1 of method of testing, recording the epoxy supported quantity is 85.2 μ mol/g.
1-3-4 takes by weighing 1g1, and the plexiglass that the 5-pentanediamine is modified adds the 2.5ml epoxychloropropane, and 4ml concentration is 0.55N NaOH, and 40 ℃ were reacted 6 hours, made epoxy chloropropionate alkanisation 1, the plexiglass that the 5-pentanediamine is modified.Through washing, ethanol washing, drying for standby.The same 1-3-1 of method of testing, recording the epoxy supported quantity is 83.2 μ mol/g.
1-3-5 takes by weighing 1g1, and the plexiglass that the 6-hexamethylene diamine is modified adds the 2.5ml epoxychloropropane, and 4ml concentration is 0.55N NaOH, and 40 ℃ were reacted 6 hours, made epoxy chloropropionate alkanisation 1, the plexiglass that the 6-hexamethylene diamine is modified.Through washing, ethanol washing, drying for standby.The same 1-3-1 of method of testing, recording the epoxy supported quantity is 81.4 μ mol/g.
1-4 is in the terminal primary amine groups of introducing of epoxide group
1-4-1 takes by weighing 1g1-3-1, adds 10ml ammoniacal liquor, reacts 9 hours down at 130 ℃, in the terminal primary amine groups of introducing of epoxide group, obtains the primary amine carrier ball that there is hydroxyl in the β position.Be washed till neutrality with distilled water, it is 23.6 μ mol/g that acid-base titration records primary amine groups content.
1-4-2 takes by weighing 1g1-3-2, adds 10ml ammoniacal liquor, reacts 9 hours down at 130 ℃, in the terminal primary amine groups of introducing of epoxide group, obtains the primary amine carrier ball that there is hydroxyl in the β position.Be washed till neutrality with distilled water, it is 26.1 μ mol/g that acid-base titration records primary amine groups content.
1-4-3 takes by weighing 1g1-3-3, adds 10ml ammoniacal liquor, reacts 9 hours down at 130 ℃, in the terminal primary amine groups of introducing of epoxide group, obtains the primary amine carrier ball that there is hydroxyl in the β position.Be washed till neutrality with distilled water, it is 29.5 μ mol/g that acid-base titration records primary amine groups content.
1-4-4 takes by weighing 1g1-3-4, adds 10ml ammoniacal liquor, reacts 9 hours down at 130 ℃, in the terminal primary amine groups of introducing of epoxide group, obtains the primary amine carrier ball that there is hydroxyl in the β position.Be washed till neutrality with distilled water, it is 25.1 μ mol/g that acid-base titration records primary amine groups content.
1-4-5 takes by weighing 1g1-3-5, adds 10ml ammoniacal liquor, reacts 9 hours down at 130 ℃, in the terminal primary amine groups of introducing of epoxide group, obtains the primary amine carrier ball that there is hydroxyl in the β position.Be washed till neutrality with distilled water, it is 21.4 μ mol/g that acid-base titration records primary amine groups content.
1-4-6 takes by weighing 1g1-3-1, adds 10ml1, and the 2-ethylenediamine reacted 9 hours down at 130 ℃, at the terminal ethylenediamine group of introducing of epoxide group.Be washed till neutrality with distilled water, it is 63.1 μ mol/g that acid-base titration records primary amine groups content.
1-4-7 takes by weighing 1g1-3-2, adds 10ml1, and the 2-ethylenediamine reacted 9 hours down at 130 ℃, at the terminal ethylenediamine group of introducing of epoxide group.Be washed till neutrality with distilled water, it is 67.5 μ mol/g that acid-base titration records primary amine groups content.
1-4-8 takes by weighing 1g1-3-3, adds 10ml1, and the 2-ethylenediamine reacted 9 hours down at 130 ℃, at the terminal ethylenediamine group of introducing of epoxide group.Be washed till neutrality with distilled water, it is 65.2 μ mol/g that acid-base titration records primary amine groups content.
1-4-9 takes by weighing 1g1-3-4, adds 10ml1, and the 2-ethylenediamine reacted 9 hours down at 130 ℃, at the terminal ethylenediamine group of introducing of epoxide group.Be washed till neutrality with distilled water, it is 70.4 μ mol/g that acid-base titration records primary amine groups content.
1-4-10 takes by weighing 1g1-3-5, adds 10ml1, and the 2-ethylenediamine reacted 9 hours down at 130 ℃, at the terminal ethylenediamine group of introducing of epoxide group.Be washed till neutrality with distilled water, it is 62.8 μ mol/g that acid-base titration records primary amine groups content.
1-5 is converted into tertiary amine with primary amine
1-5-1 takes by weighing the 1g1-4-1 product, adds 10ml formic acid and 7.5ml formaldehyde, is heated to little boiling, and does not emerge to there being bubble, stops reaction.Through washing, ethanol washing, drying for standby.Make the dimethyl amine endotoxin absorbent that there is hydroxyl in the β position.It is 17.9 μ mol/g that acid-base titration records tertiary amine groups content.
1-5-2 takes by weighing the 1g1-4-2 product, adds 10ml formic acid and 7.5ml formaldehyde, is heated to little boiling, and does not emerge to there being bubble, stops reaction.Through washing, ethanol washing, drying for standby.Make the dimethyl amine endotoxin absorbent that there is hydroxyl in the β position.It is 18.1 μ mol/g that acid-base titration records tertiary amine groups content.
1-5-3 takes by weighing the 1g1-4-3 product, adds 10ml formic acid and 7.5ml formaldehyde, is heated to little boiling, and does not emerge to there being bubble, stops reaction.Through washing, ethanol washing, drying for standby.Make the dimethyl amine endotoxin absorbent that there is hydroxyl in the β position.It is 15.8 μ mol/g that acid-base titration records tertiary amine groups content.
1-5-4 takes by weighing the 1g1-4-4 product, adds 10ml formic acid and 7.5ml formaldehyde, is heated to little boiling, and does not emerge to there being bubble, stops reaction.Through washing, ethanol washing, drying for standby.Make the dimethyl amine endotoxin absorbent that there is hydroxyl in the β position.It is 21.4 μ mol/g that acid-base titration records tertiary amine groups content.
1-5-5 takes by weighing the 1g1-4-5 product, adds 10ml formic acid and 7.5ml formaldehyde, is heated to little boiling, and does not emerge to there being bubble, stops reaction.Through washing, ethanol washing, drying for standby.Make the dimethyl amine endotoxin absorbent that there is hydroxyl in the β position.It is 13.6 μ mol/g that acid-base titration records tertiary amine groups content.
1-5-6 takes by weighing the 1g1-4-6 product, adds 10ml formic acid and 7.5ml formaldehyde, is heated to little boiling, and does not emerge to there being bubble, stops reaction.Through washing, ethanol washing, drying for standby.It is 63.0 μ mol/g that acid-base titration records tertiary amine groups content.
1-5-7 takes by weighing the 1g1-4-7 product, adds 10ml formic acid and 7.5ml formaldehyde, is heated to little boiling, and does not emerge to there being bubble, stops reaction.Through washing, ethanol washing, drying for standby.It is 57.1 μ mol/g that acid-base titration records tertiary amine groups content.
1-5-8 takes by weighing the 1g1-4-8 product, adds 10ml formic acid and 7.5ml formaldehyde, is heated to little boiling, and does not emerge to there being bubble, stops reaction.Through washing, ethanol washing, drying for standby.It is 60.3 μ mol/g that acid-base titration records tertiary amine groups content
1-5-9 takes by weighing the 1g1-4-9 product, adds 10ml formic acid and 7.5ml formaldehyde, is heated to little boiling, and does not emerge to there being bubble, stops reaction.Through washing, ethanol washing, drying for standby.It is 56.2 μ mol/g that acid-base titration records tertiary amine groups content
1-5-10 takes by weighing the 1g1-4-10 product, adds 10ml formic acid and 7.5ml formaldehyde, is heated to little boiling, and does not emerge to there being bubble, stops reaction.Through washing, ethanol washing, drying for standby.It is 59.1 μ mol/g that acid-base titration records tertiary amine groups content
The preparation of 2-1 chitosan resin
To 5% shitosan aqueous acetic acid (5g shitosan+3ml glacial acetic acid+100ml water), adding is dissolved with in the 300ml toluene solution of 1g Span 80, stirs 1.5 hours.Slowly Dropwise 5 0ml 50% glutaraldehyde water solution is warming up to 50 ℃, constant temperature 1.0 hours.Add sodium hydrate aqueous solution, the pH of conditioned reaction system is an alkalescent.Be warming up to 80 ℃, constant temperature 4 hours.Use the absolute ethyl alcohol extract product, washing is weighed, and screening collection cut size size is the carrier of 280～900 μ m.Use NaBH under the room temperature alkali condition
4Reductase 12 4h cleans standby.Recording the amino content of residue with acid-base titration is 101.3～120.7 μ mol/g.
The activation of 2-1 epoxy
The 1g chitosan resin adds the 5ml methyl-sulfoxide, the NaOH of 2ml2M, and the 3ml epoxychloropropane, 40 ℃ were reacted 4 hours.Obtain the epoxy activated carrier, be washed with water to neutrality, and the thorough remaining epoxychloropropane of flush away.The supported quantity of measuring epoxy with the 1-3-1 method is 112.6 μ mol/g.
2-3 is in the terminal primary amine groups of introducing of epoxide group
2-3-1 takes by weighing 1g epoxy chloropropionate alkanisation product, adds 10ml ammoniacal liquor, reacts 9 hours down at 130 ℃, in the terminal primary amine groups of introducing of epoxide group, obtains the primary amine carrier ball that there is hydroxyl in the β position.With determination of acid-basetitration primary amine groups content is 35.6 μ mol/g.
2-3-2 takes by weighing 1g epoxy chloropropionate alkanisation product, adds 10ml1, and the 2-ethylenediamine reacted 9 hours down at 130 ℃, at the terminal ethylenediamine group of introducing of epoxide group.With determination of acid-basetitration primary amino radical content is 94.2 μ mol/g.
2-4 is converted into tertiary amine with primary amine
2-4-1 takes by weighing the 1g2-3-1 product, adds 10ml formic acid and 7.5ml formaldehyde, is heated to little boiling, and does not emerge to there being bubble, stops reaction.Through washing, ethanol washing, drying for standby.Make the dimethyl amine endotoxin absorbent that there is hydroxyl in the β position.With determination of acid-basetitration tertiary amine groups content is 30.2 μ mol/g.
2-4-2 takes by weighing the 1g2-3-2 product, adds 10ml formic acid and 7.5ml formaldehyde, is heated to little boiling, and does not emerge to there being bubble, stops reaction.Through washing, ethanol washing, drying for standby.With determination of acid-basetitration tertiary amine groups content is 89.6 μ mol/g.
Embodiment 3
The preparation of the spherical agar carrier of 3-1
The 500ml there-necked flask is placed 60 ℃ of water-baths, in bottle, add 100ml toluene, the 50ml carbon tetrachloride, the 0.5ml Tween 80 evenly stirs.Take by weighing 4 gram agar powders and add distilled water 30ml, heat fused is poured the agar of fusing in toluene-carbon tetrachloride system into, stirs, and makes agar solution be dispersed into the drop of suitable size in organic facies.Cool to room temperature is toppled over and the upper strata organic solvent, and the screening particle size is the agar ball of 280～900 μ m, after washing with water, moves into conical flask, and 4 ℃ of hygrometric states are preserved.
The activation of 3-2 epoxychloropropane
Get agar ball 20.0 grams, the NaOH that adds 29.6 milliliters of 2M, the epoxychloropropane that adds 13.3 milliliters again, reacted 2 hours down at 40 ℃, be washed with water to neutrality, and the thorough remaining epoxychloropropane of flush away, obtain the gel ball that epoxy activates, recording the epoxy radicals supported quantity with the 1-3-1 method of testing is 110.2 μ mol/g gel balls.
3-3 is in the terminal primary amine groups of introducing of epoxide group
3-3-1 takes by weighing 1g epoxy chloropropionate alkanisation product, adds 10ml ammoniacal liquor, reacts 9 hours down at 130 ℃, in the terminal primary amine groups of introducing of epoxide group, obtains the primary amine carrier ball that there is hydroxyl in the β position.Primary amine groups content is 33.5 μ mol/g.
3-3-2 takes by weighing 1g epoxy chloropropionate alkanisation product, adds 10ml1, and the 2-hexamethylene diamine reacted 9 hours down at 130 ℃, at the terminal hexamethylene diamine group of introducing of epoxide group.Primary amine groups content is 78.4 μ mol/g.
3-4 is converted into tertiary amine with primary amine
3-4-1 takes by weighing the 1g3-3-1 product, adds 10ml formic acid and 7.5ml formaldehyde, is heated to little boiling, and does not emerge to there being bubble, stops reaction.Through washing, ethanol washing, drying for standby.Make the dimethyl amine endotoxin absorbent that there is hydroxyl in the β position.Tertiary amine groups content is 27.9 μ mol/g.
3-4-2 takes by weighing the 1g3-3-2 product, adds 10ml formic acid and 7.5ml formaldehyde, is heated to little boiling, and does not emerge to there being bubble, stops reaction.Through washing, ethanol washing, drying for standby.Tertiary amine groups content is 71.2 μ mol/g.
Embodiment 4
The preparation of 4-1 ball shaped cellulose carrier
Cotton linter is joined in the aqueous sodium hypochlorite solution of 8 times of (weight ratios) 1%, regulate PH=7～8 with hydrochloric acid solution, Dropwise 5 % urea, cotton linter is bleached, and washing is extracted.Soaking at room temperature is 2 hours in the sodium hydroxide solution of cotton linter 19% of bleaching, extracts, and puts into wide-mouth bottle, adds a cover, and places under the room temperature aging three days.Get sample, the acid neutralization, actual dry weight is calculated in the oven dry of washing back.The carbon disulfide (carbon disulfide V=alkali cotton dry weight g/2) that adds amount of calculation in the alkali cotton after aging, sealing, 25 ℃ vibrated 5 hours, and obtained orange red thick liquid.According to the cotton dry weight of alkali, dilute with 6% sodium hydroxide solution that to obtain concentration be 6～12% viscose.
In the 500ml there-necked flask, add chlorobenzene 160ml, carbon tetrachloride 40ml, potassium oleate 0.4 gram, stir about 30 minutes is to evenly.Take by weighing the viscose of 60 gram absorbent cotton, add a certain amount of 300 order CaCO
3Powder, and stir.Regulate mixing speed, viscose is slowly added there-necked flask, make viscose be dispersed into the drop of suitable size with glass funnel.Constant speed stirred more than 30 minutes under the room temperature, observed the drop deployment conditions.With the speed of the about 2 ℃/min bath temperature that slowly raises, rise to 90 ℃ of insulations 2.5 hours.Remove water bath with thermostatic control, stir down and naturally cool to room temperature, topple over and the upper strata organic solvent, distinguish the flavor of to there being chlorobenzene with the carrier that a large amount of running water flushings are synthetic, wash several times with distilled water, the carrier that filters out particle size and be 280～900 μ m is standby.
The activation of 4-2 epoxychloropropane
Get 10% gel carrier that 1g drains, add NaOH and the 1.0ml epoxychloropropane of 2.0ml3.0M, 40 ℃ of shaking table vibrations were reacted 2.5 hours down.Be washed with water to neutrality, and the thorough remaining epoxychloropropane of flush away, obtaining the gel ball that epoxy activates, the same 1-3-1 of method of testing, epoxy radicals supported quantity are 82 μ mol/g gel balls.
4-3 is in the terminal primary amine groups of introducing of epoxide group
4-3-1 takes by weighing 1g epoxy chloropropionate alkanisation product, adds 10ml ammoniacal liquor, reacts 9 hours down at 130 ℃, in the terminal primary amine groups of introducing of epoxide group, obtains the primary amine carrier ball that there is hydroxyl in the β position.With determination of acid-basetitration primary amine groups content is 30.5 μ mol/g.
4-3-2 takes by weighing 1g epoxy chloropropionate alkanisation product, adds 10ml1, and the 2-hexamethylene diamine reacted 9 hours down at 130 ℃, at the terminal hexamethylene diamine group of introducing of epoxide group.With determination of acid-basetitration primary amine groups content is 62.4 μ mol/g.
4-4 is converted into tertiary amine with primary amine
4-4-1 takes by weighing the 1g4-3-1 product, adds 10ml formic acid and 7.5ml formaldehyde, is heated to little boiling, and does not emerge to there being bubble, stops reaction.Through washing, ethanol washing, drying for standby.Make the dimethyl amine endotoxin absorbent that there is hydroxyl in the β position.With determination of acid-basetitration tertiary amine groups content is 22.6 μ mol/g.
4-4-2 takes by weighing the 1g4-3-2 product, adds 10ml formic acid and 7.5ml formaldehyde, is heated to little boiling, and does not emerge to there being bubble, stops reaction.Through washing, ethanol washing, drying for standby.With determination of acid-basetitration tertiary amine groups content is 53.2 μ mol/g.
Embodiment 5
The blood compatibility experiment
Get each 2 milliliters in the adsorbent of embodiment 1,2,3 preparation respectively, in physiological saline, soak in the perfusion post of packing into after 12 hours, inject 5 milliliters of rabbit whole bloods that are mixed with anti-coagulants, with the flow velocity perfusion of 15ml/min 2 hours with syringe.Simultaneously with empty perfusion post post in contrast.Measure the variation of perfusion front and back each composition of blood by the MEK-6318K cellanalyzer.The result shows that before and after the perfusion, various blood constituents change little, and the percentage number average of decline is in 5%, and this shows that this series adsorbent has blood compatibility preferably, can be used for whole blood perfusion.
Embodiment 6
The relation of endotoxic initial concentration and adsorbance
6-1 takes by weighing 0.050g1-5-5 adding 1ml and contains in the endotoxic aqueous solution of 10EU, and concussion 15min detects absorption front and back endotoxin content with dynamic turbidimetric.Calculating attached dose of adsorbance is 110EU/g.
6-2 takes by weighing 0.050g1-5-10 adding 1ml and contains in the endotoxic aqueous solution of 117EU, and concussion 15min detects absorption front and back endotoxin content with dynamic turbidimetric.Calculating attached dose of adsorbance is 1142EU/g.
Can see that from above-mentioned example on the basis that the adsorbent consumption is fixed (being 0.050g), along with the increase of endotoxin initial concentration, the adsorbance of unit adsorbent also is doubled and redoubled even exponential increase.This is because when endotoxin concns is big, electronegative phosphate groups on the adsorbed endotoxin molecule of adsorbent can be " bridge " by the cation in the solution, with the endotoxin molecular association in the solution, form a big molecule blastophore, this has increased the apparent adsorption capacity [Seydel of adsorbent virtually, U.et al, Immunobiology 1993.187,191-211].Therefore have the adsorbent of strong adsorption capacity under high concentration, the adsorption capacity under low concentration is not necessarily strong.Endotoxin concns is lower in the endotoxemia blood samples of patients, generally is lower than 1EU/ml.In order to investigate the adsorption capacity of adsorbent, our adsorption test is carried out under low endotoxin concn.Aqueous phase endotoxin initial concentration is decided to be 10EU/ml, and the middle mutually endotoxin initial concentration of blood is lower than 3EU/ml.(concentration is low again, and error is bigger).
Embodiment 7
The endotoxic adsorption experiment of aqueous phase
7-1 takes by weighing 0.050g1-5-5 adding 1ml and contains in the endotoxic aqueous solution of 10EU, and concussion 15min detects absorption front and back endotoxin content with dynamic turbidimetric.Calculating attached dose of adsorbance is 110EU/g, and adsorbent tertiary amine groups content 14.0 μ mo/g, so every μ mol part adsorbance is 7.84EU.
7-2 takes by weighing 0.05g1-5-10 adding 1ml and contains in the endotoxic aqueous solution of 10EU, and concussion 15min detects absorption front and back endotoxin content with dynamic turbidimetric.Calculating the adsorbents adsorb amount is 73 EU/g, and adsorbent tertiary amine groups content 59.1 μ mo/g, so every μ mol part adsorbance is 1.24EU.
Endotoxic adsorption experiment in the animal blood plasma
7-3 takes by weighing 0.050 gram chitin carrier adsorbent, adds the blood plasma of 1 milliliter of endotoxemia model big white mouse, room temperature vibration 20 minutes, endotoxin concns before and after the employing azo development process detects and adsorbs.Endotoxin concns 0.479EU/ml in the blood plasma before the absorption, absorption back 0.117EU/ml, clearance rate 75.6%.(endotoxin concns is 0.18～0.22EU/ml in the normal big white mouse blood plasma.)
7-4 takes by weighing 0.100 gram chitin carrier 1-5-3 adsorbent, adds the blood plasma of 0.400 milliliter of endotoxemia model White Rabbit, room temperature vibration 20 minutes, endotoxin concns before and after the employing azo development process detects and adsorbs.Endotoxin concns 2.58EU/ml in the blood plasma before the absorption, absorption back 0.57EU/ml.Clearance rate 77.9%.
Embodiment 8
We adopt sybyl 6.3 softwares (Tripos company) to make up β position nucleophilic group model, it is carried out molecular mechanics calculate, and the Tripos field of force is adopted in the field of force.All calculate the uncle of Nankai University and finish on the building 507 Room SGI Indigo II work stations age.Selected parameters in the calculating unless specialize, all adopts default value.Computer simulation results sees Table 1.We select tertiary amine group as terminal functionality, and the influence of the hydroxyl of the diverse location that is positioned at terminal functionality to endotoxin absorption has been discussed.Because hydrogen bond has directionality and saturability, the hydroxyl oxygen atom that only is positioned at the β position of end group just can form hydrogen bond with the hydroxyl hydrogen atom on the phosphate radical.
Table 1: the formation situation of computer simulation hydroxyl hydrogen bond when diverse location
Oxygen atom position, hydroxy position hydrogen atom position hydrogen bond hydrogen bond length
The α position can not form on adsorbent on the endotoxin molecule
On the endotoxin molecule, can not form on the adsorbent
The β position can form 0.978 on adsorbent on the endotoxin molecule
On the endotoxin molecule, can not form on the adsorbent
The γ position can not form on adsorbent on the endotoxin molecule
On the endotoxin molecule, can not form on the adsorbent
Claims (9)
1, a kind of endotoxin absorbent is characterized in that it is is carrier with spherical natural polymer or synthetic high polymer, and by the adsorbent that the arm linking ligand constitutes, the amido content of adsorbent is 10-130 μ mol/g adsorbent;
Described part is a dimethyl amine; Described arm is a saturated aliphatic chain; Described ball type carrier is shitosan, agar, cellulose or polymethacrylates.
2,, it is characterized in that the β position of described dimethyl amine is connected with hydroxyl according to the described endotoxin absorbent of claim 1.
3,, it is characterized in that described ball type carrier is a polymethyl methacrylate according to the described endotoxin absorbent of claim 1.
4,, it is characterized in that described saturated aliphatic chain is a diamines according to the described endotoxin absorbent of claim 1.
5, according to the described endotoxin absorbent of claim 4, it is characterized in that described diamines, be ethylenediamine, propane diamine, butanediamine, pentanediamine or hexamethylene diamine.
6, the preparation method of the described endotoxin absorbent of claim 1 is characterized in that:
The following step of preparation method's process of the endotoxin absorbent that spherical polymethyl methacrylate is a carrier:
(1) the spherical polymethyl methacrylate of carrier is synthetic
In methyl methacrylate that contains 0.1～0.5g benzoyl peroxide (initator) and divinylbenzene mixed liquor, join the aqueous phase that contains 10～25% common salts and 0.1～0.6% gelatin, add several 0.05～0.2% methine orchids, stir, be warming up to 50～70 ℃, polymerization 1～3 hour; Be warming up to 72～78 ℃ again, polymerization 1～3 hour; Be warming up to 82～89 ℃ again, polymerization 1～3 hour; Be warming up to 92～98 ℃ again, constant temperature 4～6 hours;
(2) introduce arm
Make solvent with DMF, plexiglass and excessive diamine reactant reacted 7～10 hours down at 100～150 ℃;
(3) epoxychloropropane activation
Add excessive epoxychloropropane and sodium hydroxide solution in the above-mentioned product, swelling is spent the night, and reacts 4～6 hours down at 25～60 ℃, makes the epoxychloropropane activation products;
(4) introduce primary amine groups
Epoxychloropropane activation products and excessive ammonia or diamine reactant reacted 7～10 hours down at 100～150 ℃, and the product that obtains connects primary amine groups at the epoxychloropropane end;
(5) primary amino radical is changed into tertiary amine groups
Add excessive formic acid and formaldehyde, be heated to boiling, do not emerge to there being bubble, through washing, ethanol washing, drying obtains the adsorbent that end group is a dimethyl amine;
The spherical shell glycan is that the preparation method of the endotoxin absorbent of carrier is through following step:
(1) spherical chitin carrier is synthetic
The shitosan aqueous acetic acid, adding is dissolved with in the toluene solution of 0.5～2.5g Span 80, stirs 0.5～2.5 hour, slowly drip 20～60ml glutaraldehyde water solution, be warming up to 35～55 ℃, constant temperature 0.5～1.5 hour, add sodium hydrate aqueous solution, the pH of conditioned reaction system is an alkalescent, is warming up to 70～85 ℃, constant temperature 3～5 hours, use the absolute ethyl alcohol extract product, washing is weighed, and the carrier of different sizes is collected in screening.Use NaBH under the room temperature alkali condition
4Reduction 12～36h cleans standby;
(2) epoxychloropropane activated carrier
Add excessive epoxychloropropane, methyl-sulfoxide and sodium hydroxide solution in the above-mentioned product, swelling is spent the night, and reacts 4～6 hours down for 60 ℃ at 251～5% shitosan aqueous acetic acids, makes the epoxychloropropane activation products;
Each step synthetic method is with polymethyl methacrylate carrier (3), (4) step later on;
The preparation method of the endotoxin absorbent of spherical agar carrier is through following step:
(1) the spherical agar carrier of preparation:
Aqueous agar solution heat fused with 2～10%, pour in toluene-carbon tetrachloride system, add Tween 80, stir, make agar solution in organic facies, be dispersed into drop, 60～90 ℃ were reacted 2～4 hours, after the cooling, topple over and the upper strata organic solvent, the agar ball of screening 0.45～1.0mm, after water fully washs, as adsorbing agent carrier;
(2) epoxychloropropane activated carrier:
Carrier in 2M NaOH solution, 30～50 ℃ down with epichlorohydrin reaction 1～4 hour, wash with water to neutrality, remove unreacted epoxychloropropane, obtain the carrier of epoxychloropropane modification;
Each step synthetic method is with polymethyl methacrylate carrier (3), (4) step later on;
The preparation method of the endotoxin absorbent of ball shaped cellulose carrier is through following step:
(1) the plain carrier of dimension is synthetic
Cotton linter is at first drifted with 1% aqueous sodium hypochlorite solution, puts into 19% sodium hydroxide solution again, and soaking at room temperature 2 hours is extracted, sealing was worn out three days under the room temperature, got sample and measured dry weight, with the carbon disulfide that measures, 25 ℃ were reacted 5 hours down, obtain orange red thick liquid then; With the chlorobenzene is decentralized medium, regulate decentralized medium proportion with carbon tetrachloride, potassium oleate is a dispersant, add calcium carbonate powder in the viscose as pore-foaming agent, adopt suspension polymerization, 80～95 ℃ were reacted 1～3 hour, washing, remove pore-foaming agent with the saturated calcium chloride solution of 0.5N hydrochloric acid (including 20% sodium chloride) hydrolysis, be washed to neutrality, the screening different meshes is standby;
Each step synthetic method is with agar carrier (2), (3) and (4) step later on.
7,, it is characterized in that the material proportion in each step according to the preparation method of the described endotoxin absorbent of claim 6:
The material proportion of spherical each step of polymethyl methacrylate preparation of adsorbent method is: methyl methacrylate: divinylbenzene (W: be 3～5: 7 W); Plexiglass: diamines (W: be 1: 5～15 V); Introduce the carrier of arm: epoxychloropropane (W: V) be: 1: 5～20; Concentration of sodium hydroxide solution is 0.55mol/L; Epoxy activated carrier: ammonia or diamines (W: be 1: 5～15 V); The carrier of primary amine groups: formic acid: formaldehyde (W: V: V) be: 1: 5～15: 5～15;
The material proportion of spherical each step of preparation method of chitosan absorbent is: shitosan: acetic acid: water (W: V: be 1～5 V): 3: 100; Chitin carrier: epoxychloropropane: methyl-sulfoxide (W: V: be 1: 1～5: 5～10 V), NaOH 2M; Epoxy activated carrier: ammonia or diamines (W: be 1: 5～15 V); The carrier of primary amine groups: formic acid: formaldehyde (W: V: V) be: 1: 5～15: 5～15;
The material proportion of each step of preparation method of spherical agar endotoxin absorbent is: toluene: carbon tetrachloride (V: V) be 1～4: 1 (ml: ml); The NaOH of agar ball: 2M: epoxychloropropane (W: V: be 1: 1～4: 0.5～2 V); Epoxy activated carrier: ammonia or diamines (W: be 1: 5～15 V); The carrier of primary amine groups: formic acid: formaldehyde (W: V: V) be: 1: 5～15: 5～15;
The material proportion of each step of preparation method of ball shaped cellulose endotoxin absorbent is: 75～100% (W: W) chlorobenzene, 0～25% (W: W) carbon tetrachloride, 0.2% (W: W) potassium oleate, form organic decentralized photo; Take by weighing the viscose of organic facies 1/3 weight, (W: calcium carbonate powder W) is as pore-foaming agent to wherein adding 0～10%; The NaOH of cellulose balls: 2M: epoxychloropropane (W: V: be 1: 1～4: 0.5～2 V); Epoxy activated carrier: ammonia or diamines (W: be 1: 5～15 V); The carrier of primary amine groups: formic acid: formaldehyde (W: V: V) be: 1: 5～15: 5～15.
8, the described endotoxin absorbent of a kind of use claim 1 is removed endotoxic method from biological products, it is characterized in that: under the room temperature, adsorbent is contained in the endotoxic biological products solution direct the adding, vibration absorption 15 minutes, adsorbent: solution (W: V) be 1: 20, detect absorption front and back endotoxin concns with dynamic method.
9, a kind of method of using the described endotoxin absorbent of claim 1 to carry out blood perfusion, it is characterized in that it comprises the steps: under the room temperature, getting adsorbent packs in the perfusion post, endotoxemia patient's whole blood is with the flow velocity perfusion of 15ml/min 2 hours, adopts azo development process to detect endotoxin concns before and after the absorption; Or under the room temperature, adsorbent is contained in the endotoxic blood plasma static absorption 20 minutes direct the adding.
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