阅读说明：本技术 一种双氨基壳聚糖-氧化石墨烯杂化材料的制备方法 (Preparation method of chitosan-graphene oxide hybrid material ) 是由 谢颖 储震宇 金万勤 于 2021-08-16 设计创作，主要内容包括：本发明涉及一种双氨基壳聚糖-氧化石墨烯杂化材料的制备方法。其制备方法包括：将制备的O-羧甲基壳聚糖与单保护的二胺类化合物在缩合剂作用下发生酸胺缩合反应后,脱除保护基,得到含有双氨基的壳聚糖衍生物；将双氨基壳聚糖聚合物与氧化石墨烯进行缩合可制备双氨基壳聚糖-石墨烯杂化材料。本发明以壳聚糖和氧化石墨烯为原料合成了双氨基壳聚糖-氧化石墨烯杂化材料,兼具有石墨烯的快速水分子通道和壳聚糖的生物相容性和亲水性,可用于构建血浆原位、即时无损分离提取的血浆分离膜,进行新鲜血液血清的快速提取,具有良好的应用前景。(The invention relates to a preparation method of a chitosan-graphene oxide hybrid material. The preparation method comprises the following steps: performing acid-amine condensation reaction on the prepared O-carboxymethyl chitosan and a mono-protected diamine compound under the action of a condensing agent, and removing a protecting group to obtain a chitosan derivative containing diamino; the diamino chitosan polymer and graphene oxide are condensed to prepare the diamino chitosan-graphene hybrid material. The chitosan-graphene oxide hybrid material is synthesized by taking chitosan and graphene oxide as raw materials, has the quick water molecular channel of graphene and the biocompatibility and hydrophilicity of chitosan, can be used for constructing a plasma separation membrane for in-situ, instant and nondestructive separation and extraction of plasma, can be used for quickly extracting fresh blood serum, and has good application prospect.)

1. A preparation method of a chitosan-graphene oxide hybrid material is characterized by comprising the following steps:

(1) dissolving O-carboxymethyl chitosan in 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution of EDC/NHS to obtain solution A, and stirring in ice bath for reaction;

(2) mixing the reaction solution obtained in the step (1) with MES buffer solution of mono-tert-butoxycarbonyl protected diamine to obtain solution B, adding sodium hydroxide to control the pH value to be 6-8, and stirring at room temperature for reaction;

(3) adding hydrochloric acid to adjust the pH value of the solution to 4.9-5.1, filtering and washing to obtain the monoamino chitosan derivative protected by the mono-tert-butoxycarbonyl group;

(4) dispersing the mono-tert-butoxycarbonyl protected diamino chitosan derivative in methanol, slowly dripping concentrated hydrochloric acid, stirring for reaction, dialyzing in deionized water, and freeze-drying to obtain diamino chitosan derivative;

(5) mixing the butyl acetate suspension of the graphene oxide with a condensing agent, and uniformly mixing the butyl acetate suspension of the graphene oxide and the condensing agent by ultrasonic waves to obtain a mixed solution C;

(6) adding the diamino chitosan derivative into the mixed solution C, and stirring at controlled temperature for reaction; and after the reaction is finished, centrifuging the reaction solution, circularly washing with deionized water, ethanol and acetone, and then drying to obtain the chitosan-graphene oxide hybrid material.

2. The preparation method of the chitosan-graphene oxide hybrid material as claimed in claim 1, wherein in the solution A in the step (1), the concentration of O-carboxymethyl chitosan is 0.01-0.05M, and the concentration of MES buffer solution of EDC/NHS is 0.01-0.2M; the stirring speed of the ice bath is 100-1000rpm, and the reaction time of the ice bath is 1-1.5 h.

3. The method for preparing a chitosan-graphene oxide hybrid material according to claim 1, wherein the concentration of the mono-tert-butoxycarbonyl protected diamine in the solution B in the step (2) is 0.01-0.2M, and the diamine is any one of N-Boc-ethylenediamine, N-Boc-1, 3-propylenediamine, N-Boc-1, 2-phenylenediamine, N-Boc-1, 6-hexamethylenediamine or N-Boc-1, 4-butanediamine; the stirring speed is 200-1000rpm at room temperature, and the reaction time is 12-36 h.

4. The preparation method of the chitosan-graphene oxide hybrid material according to claim 1, wherein the solute of the washing solution in the step (3) is any one of sodium carbonate, sodium bicarbonate, potassium carbonate or potassium bicarbonate.

5. The preparation method of the chitosan-graphene oxide hybrid material according to claim 1, wherein concentrated hydrochloric acid is added dropwise after the solution temperature is controlled to be 0-10 ℃ in the step (4); the stirring reaction temperature is 5-35 ℃, the stirring reaction time is 8-48h, and the stirring speed is 200-1000 rpm; dialyzing for 2-5 days at a temperature of- (10-20) deg.C; the freeze drying time is 1-4 days.

6. The preparation method of the chitosan-graphene oxide hybrid material according to claim 1, wherein the condensing agent in the step (5) is any one of EDC, DCC or DIC, the ultrasonic duration is 15-60min, and the ultrasonic temperature is 15-50 ℃.

7. The preparation method of the diamino chitosan-graphene oxide hybrid material as claimed in claim 1, wherein the reaction temperature after the diamino chitosan derivative is added in the step (6) is 100-; washing with deionized water and centrifuging for 1-3 times, washing with ethanol and centrifuging for 1-3 times, and washing with acetone and centrifuging for 1-3 times; the volume of the washing liquid used for each washing is 30-60 ml; the drying temperature is 30-70 ℃, and the drying time is 12-48 h; the molar ratio of the diamino chitosan derivative to the graphene oxide is (1-3): 1, and the molar ratio of the diamino chitosan derivative to the condensing agent is 1: (1-2).

Technical Field

The invention belongs to the technical field of blood separation membrane preparation, and relates to a preparation method of a chitosan-graphene oxide hybrid material.

Background

In clinical operation, autologous blood transfusion has become a research hotspot of 'blood management', the currently generally adopted autologous blood separation and purification technology is a centrifugal separation method, hemolysis is easy to generate, and the separation precision and efficiency are poor. The blood components are complex, and in order to obtain purified plasma, a membrane material needs to be 1, and the blood compatibility is good; avoid the cell damage and the hemolysis. 2. The surface is electronegative; most of proteins and cells in blood have electronegativity, and the surface of the separation membrane must have electronegativity to avoid adsorption of the proteins and cells. Chitosan has good film forming property, is nontoxic, has good biocompatibility and hydrophilicity, but is easy to generate protonation due to the existence of high-activity amino groups in molecules, so that the positive charge of a whole macromolecular chain belt can aggravate the adsorption and pore blocking of protein in blood, and is not beneficial to the implementation of instant long-term blood separation in clinic. The existing blood separation membrane preparation materials are deficient, and the preparation of a proper blood separation membrane material is a research and development hotspot and difficulty in the field at present.

Disclosure of Invention

The invention provides a preparation method of a novel diamino chitosan-graphene oxide hybrid material aiming at the problems in the preparation of the traditional blood separation membrane.

In order to achieve the purpose, the invention is realized by adopting the following technical scheme:

a preparation method of a diamino chitosan-graphene oxide hybrid material comprises the steps of introducing a diamino group into a chitosan molecule, introducing graphene oxide into a chitosan structure through dehydration condensation, and preparing the diamino chitosan-graphene oxide hybrid material with a specific structure.

The preparation method comprises the following specific steps:

(1) dissolving O-carboxymethyl chitosan in MES buffer solution of EDC/NHS to obtain solution A, and stirring in ice bath for reaction;

(2) mixing the reaction solution obtained in the step (1) with MES buffer solution of mono-tert-butoxycarbonyl protected diamine to obtain solution B, adding sodium hydroxide to control the pH value to be 6-8, and stirring at room temperature for reaction;

(3) adding hydrochloric acid to adjust the pH value of the solution to 4.9-5.1, filtering and washing to obtain the monoamino chitosan derivative protected by the mono-tert-butoxycarbonyl group;

(4) dispersing the mono-tert-butoxycarbonyl protected diamino chitosan derivative in methanol, slowly dripping concentrated hydrochloric acid, stirring for reaction, dialyzing in deionized water, and freeze-drying to obtain diamino chitosan derivative;

(5) stirring and mixing the butyl acetate suspension of the graphene oxide with a condensing agent to obtain a mixed solution C;

(6) adding the diamino chitosan derivative into the mixed solution C, and stirring at controlled temperature for reaction; and after the reaction is finished, centrifuging the reaction solution, washing with deionized water, ethanol and acetone, and drying to obtain the chitosan-graphene oxide hybrid material.

Preferably, in the solution A in the step (1), the concentration of the O-carboxymethyl chitosan is 0.01-0.05M, and the concentration of the MES buffer solution of EDC/NHS is 0.01-0.2M; the stirring speed of the ice bath is 100-1000rpm, and the reaction time of the ice bath is 1-1.5 h.

Preferably, the concentration of the mono-tert-butoxycarbonyl protected diamine in the solution B in the step (2) is 0.01-0.2M, and the diamine is any one of N-Boc-ethylenediamine, N-Boc-1, 3-propylenediamine, N-Boc-1, 2-phenylenediamine, N-Boc-1, 6-hexanediamine or N-Boc-1, 4-butanediamine; the stirring speed is 200-1000rpm at room temperature, and the reaction time is 12-36 h.

Preferably, the solute of the washing solution in the step (3) is any one of sodium carbonate, sodium bicarbonate, potassium carbonate or potassium bicarbonate.

Preferably, after the solution temperature is controlled to be 0-10 ℃, concentrated hydrochloric acid is dripped in the solution in the step (4); the stirring reaction temperature is 5-35 ℃, the stirring reaction time is 8-48h, and the stirring speed is 200-1000 rpm; dialyzing for 2-5 days at a temperature of- (10-20) deg.C; the freeze drying time is 1-4 days.

Preferably, the condensing agent in the step (5) is any one of EDC, DCC or DIC, the ultrasonic time is 15-60min, and the ultrasonic temperature is 15-50 ℃.

Preferably, the reaction temperature after the diamino chitosan derivative is added in the step (6) is 100-; washing with deionized water and centrifuging for 1-3 times, washing with ethanol and centrifuging for 1-3 times, and washing with acetone and centrifuging for 1-3 times; the volume of the washing liquid used for each washing is 30-60 ml; the drying temperature is 30-70 ℃, and the drying time is 12-48 h; the molar ratio of the diamino chitosan derivative to the graphene oxide is (1-3): 1, and the molar ratio of the diamino chitosan derivative to the condensing agent is 1: (1-2).

The surface of the graphene oxide is electronegative, has a thin lamellar structure and a unique water molecule channel, but has cytotoxicity and is easy to cause hemolysis. If the graphene and the chitosan can be combined, the separation of blood can be effectively realized by utilizing the rapid water molecule channel and the electronegativity of the graphene and the hydrophilicity and biocompatibility of the chitosan. Therefore, the chitosan derivative with the double amino groups is synthesized by an organic synthesis method, the obtained derivative is in a compact structure with graphene oxide through chemical bonds, graphene sheet layers are connected to improve film-forming performance, and the obtained hybrid material has the hydrophilicity and biocompatibility of chitosan and the electronegativity and rapid molecular channel of graphene, and can be used for preparing blood separation membranes.

Compared with the prior art, the invention has the advantages and positive effects that:

the invention develops a preparation method of a diamino chitosan-graphene oxide hybrid material. The obtained hybrid material has a compact structure with graphene oxide through chemical bonds, and graphene sheets are connected to improve the film-forming property, so that the obtained hybrid material has the hydrophilicity and biocompatibility of chitosan, the electronegativity of graphene and a fast molecular channel, and can be used for preparing blood separation membranes.

Drawings

FIG. 1 is a flow chart of preparation of a chitosan-graphene oxide hybrid material.

Fig. 2 is a schematic structural diagram of a chitosan-graphene oxide hybrid material.

Detailed Description

In order that the above objects, features and advantages of the present invention may be more clearly understood, the present invention will be further described with reference to specific embodiments. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments of the present disclosure.

Example 1

The present example provides a method for preparing a chitosan-graphene oxide hybrid material.

(1) Synthesis of diamino chitosan derivative: o-carboxymethyl chitosan (0.01M) was dissolved in 3ml of 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution of EDC (0.01 mol/l)/NHS (0.01M), reacted in ice bath at 100rpm under stirring for 1 hour, and then mixed with 10ml of MES buffer solution of N-Boc-ethylenediamine (0.01M), and about 1M NaOH was added to control the pH of the mixed solution to 6. And (3) reacting at the stirring speed of 200rpm for 24 hours at room temperature, adjusting the pH value of the mixed solution to 5 by using HCl (hydrogen chloride) of about 1M, filtering, and washing by using a saturated sodium carbonate solution to obtain the Boc-protected bisamino chitosan derivative. Uniformly dispersing the chitosan derivative in methanol, slowly adding concentrated hydrochloric acid dropwise at 0 ℃, reacting for 8h at 25 ℃ and a stirring speed of 500rpm, dialyzing for 2 days in deionized water, and freeze-drying for 3 days at-20 ℃ to obtain the diamino chitosan derivative.

(2) And mixing the butyl acetate suspension (0.01M) of the graphene oxide with DIC (0.01M), carrying out ultrasonic treatment for 15min, adding the chitosan derivative (0.01M) into the mixed solution, and reacting at 100 ℃ for 12h at a stirring speed of 100 rpm. The obtained reaction solution is washed and centrifuged twice by 30ml of deionized water, washed and centrifuged twice by 30ml of ethanol, washed and centrifuged twice by 30ml of acetone and dried for 24 hours at 60 ℃. And obtaining the diamino chitosan-graphene oxide hybrid material.

Example 2

(1) Synthesis of diamino chitosan derivative: o-carboxymethyl chitosan (0.02M) was dissolved in 3ml of 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution of EDC (0.01M)/NHS (0.2M), reacted in ice bath at 1000rpm under stirring for 1 hour, and then mixed with 10ml of MES buffer solution of N-Boc-1, 6-hexanediamine (0.2M), and about 1M NaOH was added to control the pH of the mixed solution to 7.5. Reacting at room temperature for 24h at the stirring speed of 1000rpm, adjusting the pH value of the mixed solution to 5 by about 1M HCl, filtering, and washing by using a saturated sodium bicarbonate solution to obtain the Boc-protected bisamino chitosan derivative. Uniformly dispersing the chitosan derivative in methanol, slowly adding concentrated hydrochloric acid dropwise at 10 ℃, reacting for 2h at 35 ℃ at the stirring speed of 800rpm, dialyzing for 5 days in deionized water, and freeze-drying for 4 days at-10 ℃ to obtain the diamino chitosan derivative.

(2) Mixing a butyl acetate suspension (0.01M) of graphene oxide with DCC (0.02M), carrying out ultrasonic treatment for 15min, adding a chitosan bisamide derivative (0.02M) into the mixed solution, and reacting at 120 ℃ for 24h at a stirring speed of 600 rpm. The obtained reaction solution is washed and centrifuged once by 40ml of deionized water, washed and centrifuged once by 40ml of ethanol, washed and centrifuged once by 40ml of acetone and dried for 48 hours at 30 ℃. And obtaining the diamino chitosan-graphene oxide hybrid material.

Example 3

(1) Synthesis of diamino chitosan derivative: o-carboxymethyl chitosan (0.03M) was dissolved in 3ml of 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution of EDC (0.01M)/NHS (0.1M), reacted in ice bath at 400rpm for 1.5 hours, mixed with 10ml of N-Boc-1, 2-phenylenediamine (0.05M) MES buffer solution, and added with about 1M NaOH to control the pH of the mixed solution to 7. Reacting at room temperature for 36h at the stirring speed of 1000rpm, adjusting the pH value of the mixed solution to 5 by about 1M HCl, filtering, and washing by using a saturated potassium carbonate solution to obtain the Boc-protected bisamino chitosan derivative. Uniformly dispersing the chitosan derivative in methanol, slowly adding concentrated hydrochloric acid dropwise at 5 ℃, reacting for 48h at 5 ℃ and a stirring speed of 600rpm, dialyzing for 5 days in deionized water, and freeze-drying for 1 day at-15 ℃ to obtain the diamino chitosan derivative.

(2) Mixing a butyl acetate suspension (0.01M) of graphene oxide with EDC (0.04M), carrying out ultrasonic treatment for 15min, adding a chitosan bisaminoderivative (0.02M) into the mixed solution, and reacting at 110 ℃ for 12h at a stirring speed of 700 rpm. The obtained reaction solution is washed and centrifuged twice by 50ml of deionized water, washed and centrifuged twice by 50ml of ethanol, washed and centrifuged twice by 50ml of acetone and dried for 36 hours at 40 ℃. And obtaining the diamino chitosan-graphene oxide hybrid material.

Example 4

(1) Synthesis of diamino chitosan derivative: o-carboxymethyl chitosan (0.05M) was dissolved in 3ml of 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution of EDC (0.01M)/NHS (0.1M), reacted in ice bath at 300rpm under stirring for 1 hour, and then mixed with 10ml of MES buffer solution of N-Boc-1, 4-butanediamine (0.1M), and about 1M NaOH was added to control the pH of the mixed solution to 6.5. Reacting at the stirring speed of 600rpm for 12h at room temperature, adjusting the pH value of the mixed solution to 5 by about 1M HCl, filtering, and washing by using a saturated potassium bicarbonate solution to obtain the Boc protected bisamino chitosan derivative. Uniformly dispersing the chitosan derivative in methanol, slowly adding concentrated hydrochloric acid dropwise at 3 ℃, reacting for 36h at 20 ℃ and a stirring speed of 1000rpm, dialyzing for 5 days in deionized water, and freeze-drying for 2 days at-20 ℃ to obtain the diamino chitosan derivative.

(2) And mixing butyl acetate suspension (0.01M) of graphene oxide with DIC (0.03M), carrying out ultrasonic treatment at 60 ℃ for 60min, adding a diamino chitosan derivative (0.03M) into the mixed solution, and reacting at 130 ℃ for 2h at the stirring speed of 900 rpm. Washing and centrifuging the obtained reaction solution for three times by using 60ml of deionized water, washing and centrifuging for three times by using 60ml of ethanol, washing and centrifuging for three times by using 60ml of acetone, and drying for 48 hours at 70 ℃. And obtaining the diamino chitosan-graphene oxide hybrid material.

The alumina hollow fiber with the diameter of 2mm and one end sealed by silicone adhesive is dipped in 0.5 percent acetic acid solution of the hybrid material prepared in the embodiment 1 for 3 times, each time is 30s, the contact angle of the obtained blood separation membrane is 46 +/-2.5 degrees, and the contact angle can be 0.30 ml min^-1The rate of (a) for separation of serum.

The above description is only a preferred embodiment of the present invention, and not intended to limit the present invention in other forms, and any person skilled in the art may apply the above modifications or changes to the equivalent embodiments with equivalent changes, without departing from the technical spirit of the present invention, and any simple modification, equivalent change and change made to the above embodiments according to the technical spirit of the present invention still belong to the protection scope of the technical spirit of the present invention.