CN115254009A - Preparation method of blood perfusion device filler for reducing uric acid concentration - Google Patents
Preparation method of blood perfusion device filler for reducing uric acid concentration Download PDFInfo
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- CN115254009A CN115254009A CN202211071319.4A CN202211071319A CN115254009A CN 115254009 A CN115254009 A CN 115254009A CN 202211071319 A CN202211071319 A CN 202211071319A CN 115254009 A CN115254009 A CN 115254009A
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- 239000000945 filler Substances 0.000 title claims abstract description 33
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 title claims abstract description 29
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 title claims abstract description 29
- 229940116269 uric acid Drugs 0.000 title claims abstract description 29
- 230000008081 blood perfusion Effects 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 124
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims abstract description 73
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 54
- 239000002245 particle Substances 0.000 claims abstract description 51
- 239000000243 solution Substances 0.000 claims abstract description 43
- 108010092464 Urate Oxidase Proteins 0.000 claims abstract description 34
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000011259 mixed solution Substances 0.000 claims abstract description 29
- 229940005267 urate oxidase Drugs 0.000 claims abstract description 24
- 238000002156 mixing Methods 0.000 claims abstract description 17
- 239000007983 Tris buffer Substances 0.000 claims abstract description 14
- 239000007864 aqueous solution Substances 0.000 claims abstract description 14
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000003999 initiator Substances 0.000 claims abstract description 11
- JXWKMMCPAYWOKV-UHFFFAOYSA-N methyl propyl hydrogen phosphate Chemical compound CCCOP(O)(=O)OC JXWKMMCPAYWOKV-UHFFFAOYSA-N 0.000 claims abstract description 5
- LHJROESDWMXAKD-UHFFFAOYSA-N trihydroxysilicon Chemical compound O[Si](O)O LHJROESDWMXAKD-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 29
- 239000008367 deionised water Substances 0.000 claims description 18
- 229910021641 deionized water Inorganic materials 0.000 claims description 18
- 238000005406 washing Methods 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 13
- 235000019441 ethanol Nutrition 0.000 claims description 10
- 238000003760 magnetic stirring Methods 0.000 claims description 8
- 238000009210 therapy by ultrasound Methods 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 6
- -1 3- (trihydroxysilyl) propyl methyl Chemical group 0.000 claims description 5
- 229910019142 PO4 Inorganic materials 0.000 claims description 5
- 239000010452 phosphate Substances 0.000 claims description 5
- 230000001951 hemoperfusion Effects 0.000 claims description 4
- ICNCZFQYZKPYMS-UHFFFAOYSA-N 2-methylpropanoyl bromide Chemical compound CC(C)C(Br)=O ICNCZFQYZKPYMS-UHFFFAOYSA-N 0.000 claims description 3
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims description 3
- 229910052794 bromium Inorganic materials 0.000 claims description 3
- HDUNVICUTAZXTE-UHFFFAOYSA-N methyl(3-trihydroxysilylpropoxy)phosphinic acid Chemical compound CP(O)(=O)OCCC[Si](O)(O)O HDUNVICUTAZXTE-UHFFFAOYSA-N 0.000 claims description 2
- 238000010992 reflux Methods 0.000 claims description 2
- 238000001291 vacuum drying Methods 0.000 claims description 2
- 238000011049 filling Methods 0.000 claims 1
- 239000000377 silicon dioxide Substances 0.000 abstract description 19
- 210000004369 blood Anatomy 0.000 abstract description 7
- 239000008280 blood Substances 0.000 abstract description 7
- 235000012239 silicon dioxide Nutrition 0.000 description 20
- 239000000047 product Substances 0.000 description 18
- 238000003756 stirring Methods 0.000 description 18
- 238000001914 filtration Methods 0.000 description 10
- 239000003463 adsorbent Substances 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 238000005576 amination reaction Methods 0.000 description 3
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 230000010412 perfusion Effects 0.000 description 3
- 238000004448 titration Methods 0.000 description 3
- YOCIJWAHRAJQFT-UHFFFAOYSA-N 2-bromo-2-methylpropanoyl bromide Chemical compound CC(C)(Br)C(Br)=O YOCIJWAHRAJQFT-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 108010093096 Immobilized Enzymes Proteins 0.000 description 2
- POJWUDADGALRAB-UHFFFAOYSA-N allantoin Chemical compound NC(=O)NC1NC(=O)NC1=O POJWUDADGALRAB-UHFFFAOYSA-N 0.000 description 2
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- 229920005989 resin Polymers 0.000 description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 2
- POJWUDADGALRAB-PVQJCKRUSA-N Allantoin Natural products NC(=O)N[C@@H]1NC(=O)NC1=O POJWUDADGALRAB-PVQJCKRUSA-N 0.000 description 1
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- 208000007536 Thrombosis Diseases 0.000 description 1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
- B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3621—Extra-corporeal blood circuits
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
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- Heart & Thoracic Surgery (AREA)
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- Cardiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Anesthesiology (AREA)
- Veterinary Medicine (AREA)
- Analytical Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention provides a preparation method of a blood perfusion device filler for reducing the concentration of uric acid, which comprises the following steps: step 1, fully mixing silica particles and HCl to activate the silica particles; step 2, reacting the activated silicon dioxide particles with absolute ethyl alcohol and 3- (trihydroxy silicon base) propyl methyl phosphate to obtain aminated silicon dioxide particles; step 3, fully mixing the aminated silica particles with N, N-dimethylformamide and triethylamine to obtain a mixed solution A, adding an initiator into the N, N-dimethylformamide to obtain a mixed solution B, and uniformly mixing the mixed solution A and the mixed solution B to obtain brominated silica particles; and 4, mixing the brominated silica particles, the Tris buffer solution and the aqueous solution of the urate oxidase to obtain the modified silica particle filler with the urate oxidase fixed on the surface, so that the urate oxidase is firmly fixed on the silica filler particles, and the modified silica particle filler has the advantage of quickly and efficiently decomposing uric acid in blood.
Description
Technical Field
The invention belongs to the technical field, and particularly relates to a preparation method of a blood perfusion device filler for reducing the concentration of uric acid.
Background
Currently, blood perfusion is a blood purification technique in which blood is introduced into an perfusion device filled with a solid adsorbent, and exogenous or endogenous toxins, drugs or metabolic waste products which cannot be removed by dialysis in the blood are removed by adsorption.
Because the solid adsorbent filled inside is made of polystyrene resin or activated carbon, the solid adsorbent is mostly directly supplied by suppliers at present, so that the autonomous research and development of users are less, the types of substances adsorbed by the adsorbent are limited, and the molecular weight of the adsorbed substances cannot be accurately controlled, so that most of blood perfusion apparatuses are subjected to nonspecific adsorption. When the hemoperfusion apparatus is used, some pathogenic substances are often effectively adsorbed, and other body fluid components with similar molecular weights are also adsorbed. This can result in the loss of beneficial bodily fluid components, causing some potential risks.
At present, some users modify and reprocess the solid adsorbent to different degrees, but the filler substrate is limited to the product attribute of the resin supplier, and is difficult to flexibly select in practical clinical application. The prior adsorbent provided by most adsorbent suppliers has large batch-to-batch difference of adsorbent packing base materials, unstable biocompatibility, incomplete contact between the packing and harmful substances in blood and low adsorption efficiency. The existing modification or modification method does not fundamentally change the basic structure of the resin, the problem of non-specific adsorption is not well solved, and the corresponding filler perfusion device cannot be selected according to specific requirements, so that the application range of the perfusion device is limited.
Disclosure of Invention
The invention provides a preparation method of a blood perfusion device filler for reducing the concentration of uric acid, which solves the problems.
The technical scheme of the invention is realized as follows: a preparation method of a blood perfusion device filler for reducing uric acid concentration comprises the following steps:
step 1, fully mixing silicon dioxide particles and HCl to activate the silicon dioxide particles;
step 2, reacting the activated silicon dioxide particles with absolute ethyl alcohol and 3- (trihydroxy silicon base) propyl methyl phosphate to obtain aminated silicon dioxide particles;
step 3, fully mixing the aminated silica particles with N, N-dimethylformamide and triethylamine to obtain a mixed solution A, adding an initiator into the N, N-dimethylformamide to obtain a mixed solution B, and fully and uniformly mixing the mixed solution A and the mixed solution B to obtain brominated silica particles;
and 4, fully mixing the brominated silica particles, the Tris buffer solution and the aqueous solution of the urate oxidase to obtain the modified silica particle filler with the urate oxidase fixed on the surface.
As a preferred embodiment, the silica particles and HCl are added in step 1 in the ratio of silica particles (g): HCl (ml) =1:4;
the silica particles and HCl are thoroughly mixed in step 1 by magnetic stirring at room temperature after ultrasonic treatment.
As a preferred embodiment, the silica particles and HCl are thoroughly mixed in step 1, and then filtered and washed by using deionized water, and then stored in a 20% ethanol solution at a constant temperature of 4 ℃.
As a preferred embodiment, the activated silica particles, anhydrous ethanol, and 3- (trishydroxysilyl) propylmethylphosphonate in step 2 are added in the following ratio, activated silica particles (g): absolute ethanol (ml): 3- (trihydroxysilyl) propylmethylphosphonate (ml) =1:2 (0.6-0.8);
in the step 2, the activated silicon dioxide particles, the absolute ethyl alcohol and the 3- (trihydroxy silicon base) propyl methyl phosphate react under the condition of condensing and refluxing at the constant temperature of 4 ℃, and then the magnetic stirring is carried out.
As a preferred embodiment, after the activated silica particles are reacted with the absolute ethyl alcohol and the 3- (trihydroxysilyl) propyl methyl phosphate in the step 2, the reaction product is further filtered and washed by respectively washing the reaction product with the absolute ethyl alcohol and the deionized water, and storing the reaction product in a 20% ethanol solution at a constant temperature of 4 ℃ after washing.
As a preferred embodiment, the silica particles aminated in step 3 and N, N-dimethylformamide and triethylamine are added in such a ratio that the silica particles aminated (g): n, N-dimethylformamide (ml) triethylamine (ml) =1:3 (0.4-0.6).
As a preferred embodiment, the initiator and N, N-dimethylformamide are added in step 3 in the ratio of initiator (ml): n, N-dimethylformamide (ml) = 1.5;
the initiator is alpha-bromine isobutyryl bromide.
As a preferable embodiment, the manner of sufficiently and uniformly mixing the mixed solution a and the mixed solution B in step 3 is to dropwise add the mixed solution B into the mixed solution a under the ice bath condition, move to a constant temperature of 30 ℃ for continuous reaction after magnetic stirring reaction, and wash with N, N-dimethylformamide, absolute ethyl alcohol and deionized water respectively after the reaction is finished.
As a preferred embodiment, the addition ratio of the aqueous solution of the brominated silica particles, tris buffer and urate oxidase in step 4 is, the ratio of the brominated silica particles (g): tris buffer (ml): aqueous solution (ml) =2 of urate oxidase;
the method for fully mixing the brominated silica particles, the Tris buffer solution and the aqueous solution of the urate oxidase in the step 4 is to carry out magnetic stirring reaction at the constant temperature of 4 ℃.
As a preferable embodiment, after fully mixing the brominated silica particles, the Tris buffer solution and the aqueous solution of the urate oxidase, filtering, washing and drying are further carried out, wherein the washing mode is deionized water, and the drying mode is vacuum drying.
After the technical scheme is adopted, the invention has the beneficial effects that:
1. the modified silica filler particles with urate oxidase fixed on the surface can quickly and efficiently decompose uric acid in blood, and the adaptation disease range of the conventional blood perfusion device is expanded.
2. The silica particles adopted by the invention have no macroporous structure on the surface, so that the hemodynamics is more stable, and the thrombosis is reduced.
3. The filler matrix of the hemoperfusion apparatus is activated by utilizing the surface groups of the silicon dioxide particles, and the urate oxidase is firmly fixed on the particle surface to avoid falling off. The reaction condition is mild, so that the urate oxidase can keep the maximum enzyme activity.
4. The reaction of the filler and a single substance in blood is more efficient, the treatment time of the whole blood perfusion is shortened, the possibility of risk occurrence in the process is reduced, and the utilization rate of medical resources is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic flow chart of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.
Example 1
As shown in fig. 1, the steps of the method of the present invention include: 1. activating silicon dioxide particles; 2. amination of the silica particles; 3. brominating the silica particles; 4. the specific preparation method of the immobilized uricase comprises the following steps:
50g of silicon dioxide particles with the particle size of 0.8mm are taken to be placed in a 500ml beaker, 200ml of HCl solution with the concentration of 1mol/L is added, and the mixture is placed in an ultrasonic pool for ultrasonic treatment for 20min after being fully and uniformly stirred.
Magnetically stirring at 1200rpm for 36h at room temperature to remove impurities adsorbed on the surface of the silica particles and fully expose and activate the hydroxyl groups on the surface. Filtering after the activation treatment is finished, washing the product for 5 times by using deionized water, and placing the product of the activated silicon dioxide particles into a 20% ethanol solution with the constant temperature of 4 ℃ for storage for later use.
Putting all the activated silicon dioxide particles into a 250ml beaker, adding 100ml of absolute ethyl alcohol, fully and uniformly stirring, and then putting into an ultrasonic pool for ultrasonic treatment for 5-10min. Then 36ml of 3- (tricarboxysilyl) propyl methyl phosphate is added, the mixture is condensed and refluxed under the condition of a water bath with the constant temperature of 4 ℃, and the reaction is magnetically stirred at the rotating speed of 240rpm for 12 hours.
And (3) filtering out products after the reaction is finished, respectively washing the products for 3 times by using absolute ethyl alcohol and deionized water, removing unreacted reagents to obtain silicon dioxide particles with aminated surfaces, and placing the silicon dioxide particles in a 20% ethanol solution with constant temperature of 4 ℃ for storage and standby.
Putting all the silicon dioxide particles into a 250ml beaker, adding 150ml N, N-dimethylformamide, fully stirring uniformly, adding 24ml triethylamine into the solution, and continuing stirring for 10-15min to fully mix uniformly to obtain a mixed solution A.
Taking 12ml of initiator alpha-bromine isobutyryl bromide to a constant pressure titration funnel, adding 15ml of N, N-dimethylformamide for dilution, and shaking up to obtain a mixed solution B.
Dropwise adding the solution B into the solution A under an ice bath condition, magnetically stirring at the rotating speed of 240rpm for reaction for 2 hours, then transferring the solution into a constant-temperature water bath at the temperature of 30 ℃ for continuous reaction for 12 hours, and respectively centrifugally washing the solution for 3 times by using N, N-dimethylformamide, absolute ethyl alcohol and deionized water after the reaction is finished to obtain the brominated silicon dioxide particles.
Adding 50g of brominated silica particles into 250ml of Tris buffer solution with the pH value of 8.0, then adding 25ml of aqueous solution containing 40mg of urate oxidase, magnetically stirring at the constant temperature of 4 ℃ and the rotation speed of 120rpm for reaction for 6 hours, filtering the product after the reaction is finished, washing for 3 times, and then drying in vacuum to obtain the modified silica particle filler with urate oxidase fixed on the surface.
Example 2
As shown in fig. 1, the steps of the method of the present invention include: 1. activating silicon dioxide particles; 2. amination of the silica particles; 3. brominating the silica particles; 4. the specific preparation method of the uricase immobilized enzyme comprises the following steps:
100g of silicon dioxide particles with the particle size of 1.2mm are taken to be placed in a 1000ml beaker, 400ml of HCl solution with the concentration of 1mol/L is added, and the mixture is placed in an ultrasonic pool for ultrasonic treatment for 20min after being fully and uniformly stirred.
Magnetically stirring at 1200rpm for 36h at room temperature to remove impurities adsorbed on the surface of the silica particles and fully expose and activate the hydroxyl groups on the surface. Filtering after the activation treatment is finished, washing the product for 5 times by using deionized water, and placing the product of the activated silicon dioxide particles into a 20% ethanol solution with the constant temperature of 4 ℃ for storage for later use.
Putting all the activated silicon dioxide particles into a 500ml beaker, adding 160ml of absolute ethyl alcohol, fully and uniformly stirring, and then putting into an ultrasonic pool for ultrasonic treatment for 5-10min. Then 48ml of 3- (tricarboxysilyl) propylmethylphosphonate is added, the mixture is condensed and refluxed under the condition of a water bath with the constant temperature of 4 ℃, and the reaction is magnetically stirred at the rotating speed of 240rpm for 12 hours.
And filtering out a product after the reaction is finished, washing the product for 3 times by respectively using absolute ethyl alcohol and deionized water, removing unreacted reagents to obtain silicon dioxide particles with aminated surfaces, and placing the silicon dioxide particles in a 20% ethanol solution with the constant temperature of 4 ℃ for storage for later use.
Putting all the silicon dioxide particles into a 250ml beaker, adding 195ml of N, N-dimethylformamide, fully stirring uniformly, adding 26ml of triethylamine into the solution, and continuing stirring for 10-15min to fully mix uniformly to obtain a mixed solution A.
15.6ml of initiator alpha-bromoisobutyryl bromide is taken in a constant pressure titration funnel, 195ml of N, N-dimethylformamide is added for dilution, and the obtained mixture is shaken up to obtain a mixed solution B.
Dropwise adding the solution B into the solution A under an ice bath condition, magnetically stirring at the rotating speed of 240rpm for reaction for 2 hours, then transferring the solution into a constant-temperature water bath at the temperature of 30 ℃ for continuous reaction for 12 hours, and respectively centrifugally washing the solution for 3 times by using N, N-dimethylformamide, absolute ethyl alcohol and deionized water after the reaction is finished to obtain the brominated silicon dioxide particles.
Adding 60g of brominated silica particles into 300ml of Tris buffer solution with the pH value of 8.0, adding 30ml of aqueous solution containing 40mg of urate oxidase, magnetically stirring at the constant temperature of 4 ℃ and the rotation speed of 120rpm for reaction for 6 hours, filtering the product after the reaction is finished, washing for 3 times, and drying in vacuum to obtain the modified silica particle filler with urate oxidase fixed on the surface.
Example 3
As shown in fig. 1, the steps of the method of the present invention include: 1. activating silicon dioxide particles; 2. amination of the silica particles; 3. brominating silica particles; 4. the specific preparation method of the uricase immobilized enzyme comprises the following steps:
150g of silicon dioxide particles with the particle size of 1.2mm are taken to be placed in a 1000ml beaker, 600ml of HCl solution with the concentration of 1mol/L is added, and the mixture is placed in an ultrasonic pool for ultrasonic treatment for 20min after being fully and uniformly stirred.
Magnetically stirring at 1200rpm for 36h at room temperature to remove impurities adsorbed on the surface of the silica particles and fully expose and activate the hydroxyl groups on the surface. Filtering after the activation treatment is finished, washing the product for 5 times by using deionized water, and placing the product activated silicon dioxide particles into a 20% ethanol solution with the constant temperature of 4 ℃ for storage for later use.
Putting all the activated silicon dioxide particles into a 500ml beaker, adding 180ml of absolute ethyl alcohol, fully and uniformly stirring, and then putting into an ultrasonic pool for ultrasonic treatment for 5-10min. Then 72ml of 3- (tricarboxysilyl) propyl methyl phosphate is added, the mixture is condensed and refluxed under the condition of a water bath with the constant temperature of 4 ℃, and the reaction is magnetically stirred at the rotating speed of 240rpm for 12 hours.
And (3) filtering out products after the reaction is finished, respectively washing the products for 3 times by using absolute ethyl alcohol and deionized water, removing unreacted reagents to obtain silicon dioxide particles with aminated surfaces, and placing the silicon dioxide particles in a 20% ethanol solution with constant temperature of 4 ℃ for storage and standby.
Putting all the silicon dioxide particles into a 250ml beaker, adding 225ml of N, N-dimethylformamide, fully stirring uniformly, adding 45ml of triethylamine into the solution, and continuing stirring for 10-15min to fully mix uniformly to obtain a mixed solution A.
Taking 18ml of initiator alpha-bromoisobutyryl bromide to a constant pressure titration funnel, adding 225ml of N, N-dimethylformamide for dilution, and shaking up to obtain a mixed solution B.
Dropwise adding the solution B into the solution A under an ice bath condition, magnetically stirring at the rotating speed of 240rpm for reaction for 2 hours, then transferring the solution into a constant-temperature water bath at the temperature of 30 ℃ for continuous reaction for 12 hours, and respectively centrifugally washing the solution for 3 times by using N, N-dimethylformamide, absolute ethyl alcohol and deionized water after the reaction is finished to obtain the brominated silicon dioxide particles.
Adding 70g of brominated silica particles into 350ml of Tris buffer solution with the pH value of 8.0, adding 35ml of aqueous solution containing 40mg of urate oxidase, magnetically stirring at the constant temperature of 4 ℃ and the rotation speed of 120rpm for reaction for 6 hours, filtering the product after the reaction is finished, washing for 3 times, and drying in vacuum to obtain the modified silica particle filler with urate oxidase fixed on the surface.
Comparative example
The hemoperfusion ware includes the cylinder, and the cylinder intussuseption is filled with the filler, and the top of cylinder is provided with the entry through last lid threaded connection, and the top of going up the lid is provided with the entry, and the bottom of cylinder is provided with the export through lid threaded connection setting down, the bottom of lower lid.
In the comparative example, plasma is input into the blood perfusion device through the peristaltic pump, the plasma is output after being filtered and adsorbed, the output plasma is detected through a uric acid detection experiment, and modified silica particle fillers and common fillers are respectively placed in a column body of the blood perfusion device.
The method for detecting uric acid comprises the following steps:
uric acid has specific ultraviolet absorption at 293nm, and allantoin, a product generated after the hydrolysis of the uricase, has no specificity. Therefore, the difference value of the absorbance before and after the uricase catalyzes is in direct proportion to the concentration of the uric acid.
The reagent kit comprises:
1.0.2mol/L boric acid-NaOH buffer (pH 9.5)
2.1.9mg/ml uricase
3.5 948.4 mu mol/L uric acid standard stock solution
4.594.84 mu mol/L uric acid standard application liquid
5.UV-3000 Shimadzu dual-wavelength/dual-light-speed automatic recording spectrophotometer
The detection method comprises the following steps:
1. mu.l of serum and 3.0ml of buffer were mixed in a quartz cuvette and absorbance (A1) of uric acid at 293nm was measured in the absence of air.
2. Mu.l of uricase solution was added to the cuvette and mixed, and then absorbance of the reacted uric acid at 293nm to air was measured (A2).
3. And (3) measuring the absorbance (Ae) of the uricase, adding 3.06ml of buffer solution into each of two quartz cuvettes, adding 10 mu l of uricase solution into one cuvette, mixing the solution uniformly, and using the other cuvette as a reference cuvette to measure the absorbance of the uricase at 293 nm.
The calculation formula is as follows:
Δ a = (A1 + Ae) -A2 of sample
Wherein 0.075 is the absorbance at 293nm through an optical path of 1cm when 5.9484. Mu. Mol/L uric acid is oxidized.
The results of the plasma tests with modified filler (obtained in example 1) are shown in the following table:
the effect of the plasma detection through the common filler is shown in the following table:
from the above results, it can be seen that the plasma uric acid content is significantly reduced by the modified silica particle filler, and the modified silica particle filler has a rapid and efficient decomposition effect on uric acid in plasma.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A preparation method of a blood perfusion device filler for reducing uric acid concentration is characterized by comprising the following steps:
step 1, fully mixing silica particles and HCl to activate the silica particles;
step 2, reacting the activated silicon dioxide particles with absolute ethyl alcohol and 3- (trihydroxy silicon base) propyl methyl phosphate to obtain aminated silicon dioxide particles;
step 3, fully mixing the aminated silica particles with N, N-dimethylformamide and triethylamine to obtain a mixed solution A, adding an initiator into the N, N-dimethylformamide to obtain a mixed solution B, and fully and uniformly mixing the mixed solution A and the mixed solution B to obtain brominated silica particles;
and 4, fully mixing the brominated silica particles, the Tris buffer solution and the aqueous solution of the urate oxidase to obtain the modified silica particle filler with the urate oxidase fixed on the surface.
2. The method for preparing a hemoperfusion cartridge filling for reducing uric acid concentration according to claim 1, wherein the silica particles and HCl are added in the ratio of (g) silica particles: HCl (ml) =1:4;
the silica particles and HCl are thoroughly mixed in step 1 by magnetic stirring at room temperature after ultrasonic treatment.
3. The method for preparing a blood perfusion filler for reducing uric acid concentration as claimed in claim 1, wherein the silica particles and HCl are fully mixed in step 1, and then the mixture is filtered and washed by using deionized water, and the deionized water is washed, and then stored and placed in a 20% ethanol solution at a constant temperature of 4 ℃.
4. The method for preparing a blood perfusion filler for reducing uric acid concentration as claimed in claim 1, wherein the activated silica particles, the absolute ethyl alcohol and the 3- (trihydroxysilyl) propyl methyl phosphate in the step 2 are added in a proportion that the activated silica particles (g): absolute ethanol (ml): 3- (trihydroxysilyl) propylmethylphosphonate (ml) =1:2 (0.6-0.8);
in the step 2, the activated silicon dioxide particles, the absolute ethyl alcohol and the 3- (trihydroxy silicon base) propyl methyl phosphate react under the condition of condensing and refluxing at the constant temperature of 4 ℃, and then the magnetic stirring is carried out.
5. The method for preparing a blood perfusion filler for reducing uric acid concentration as claimed in claim 1, wherein the activated silica particles, the absolute ethyl alcohol and the 3- (trihydroxysilyl) propyl methyl phosphate react in the step 2, and then the mixture is filtered and washed by respectively adopting the absolute ethyl alcohol and the deionized water, and the washed mixture is stored and placed in a 20% ethanol solution with a constant temperature of 4 ℃.
6. The method for preparing a blood perfusion filter for reducing uric acid concentration as claimed in claim 1, wherein the addition ratio of the aminated silica particles, N-dimethylformamide and triethylamine in step 3 is as follows: n, N-dimethylformamide (ml): triethylamine (ml) =1:3 (0.4-0.6).
7. The method for preparing a blood perfusion filler for reducing uric acid concentration as claimed in claim 1, wherein the initiator and N, N-dimethylformamide are added in the proportion of (ml) in step 3: n, N-dimethylformamide (ml) = 1.5;
the initiator is alpha-bromine isobutyryl bromide.
8. The method for preparing a blood perfusion filler for reducing uric acid concentration according to claim 1, wherein the mixed solution A and the mixed solution B are sufficiently and uniformly mixed in the step 3 in such a manner that the mixed solution B is dropwise added into the mixed solution A under an ice bath condition, after magnetic stirring reaction, the mixed solution A is moved to a constant temperature of 30 ℃ for continuous reaction, and after the reaction is finished, the mixed solution A and the mixed solution B are washed with N, N-dimethylformamide, absolute ethyl alcohol and deionized water respectively.
9. The method for preparing a blood perfusion filter for reducing uric acid concentration as claimed in claim 1, wherein the adding proportion of the aqueous solution of the brominated silica particles, the Tris buffer and the urate oxidase in the step 4 is that the brominated silica particles (g): tris buffer (ml): aqueous solution (ml) =2 of urate oxidase;
the method for fully mixing the brominated silica particles, the Tris buffer solution and the aqueous solution of the urate oxidase in the step 4 is to carry out magnetic stirring reaction at the constant temperature of 4 ℃.
10. The method for preparing a blood perfusion filler for reducing uric acid concentration as claimed in claim 1, wherein the brominated silica particles, the Tris buffer solution and the aqueous solution of urate oxidase are fully mixed, and then are filtered, washed and dried, wherein the washing mode is deionized water, and the drying mode is vacuum drying.
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