Method for preparing modified cellulose aerogel for glycoprotein separation

Abstract

A method for preparing a modified cellulose aerogel for glycoprotein separation is provided. In this method, cellulose aerogel is employed as a substrate. The cellulose aerogel is known to have a three-dimensional network structure with extremely high porosity and specific surface area and extremely low density. So, by using the cellulose aerogel as a substrate, it is possible to provide the glycoproteins to be separated with more binding sites. PEI dendrimer has abundant functional groups and can easily be modified. By modifying the cellulose aerogel substrate with the PEI dendrimer, it is possible to improve the density of the phenylboronic acid bound to the substrate, thereby leading to higher affinity toward the glycoproteins to be separated.

Claims

1. A method for preparing a modified cellulose aerogel for glycoprotein separation, comprising steps of: (1) adding sodium hydroxide and urea to deionized water and stirring to form a first solution, which is pre-cooled at −12.5° C.; (2) into the pre-cooled first solution, cellulose is added and completely dissolved and then a volume of 2 to 5 mL of epichlorohydrin is added to form a second solution, which is poured into a mould and allowed to stand for 24 h and is then washed and freeze dried to obtain a regenerated cellulose aerogel; (3) adding the regenerated cellulose aerogel obtained in the step (2) and sodium periodate to deionized water and stirring to form a third solution, which is subjected to a reaction in the dark at a temperature of 50 to 70° C. for 5 to 7 h, at the end of which time a volume of 3 to 5 mL of ethylene glycol is added and stirred for 30 to 60 min followed by washing with deionized water and ethanol and then freeze drying to obtain a dialdehyde cellulose aerogel; (4) adding the dialdehyde cellulose aerogel obtained in the step (3) to anhydrous ethanol to form a fourth solution, to which polyethyleneimine (PEI) and sodium cyanoborohydride are added to conduct a reaction at a temperature of 50 to 70° C. for 6 h followed by washing with ethanol to neutral and then freeze drying to obtain a PEI-modified cellulose aerogel; and (5) dissolving 4-formylphenylboronic acid in a volume of 100 to 500 mL of anhydrous ethanol to form a fifth solution, to which the PEI-modified cellulose aerogel obtained in the step (4) and sodium cyanoborohydride are added to conduct a reaction at 50° C. for 12 to 24 h followed by washing with distilled water and ethanol and then vacuum drying to obtain a PEI/phenylboronic acid (PBA)-modified cellulose aerogel.

2. The method of claim 1, wherein, in the step (1), the sodium hydroxide, urea, and deionized water are employed in an amount of 6 to 10 g, 9 to 13 g, and 77 to 85 mL, respectively.

3. The method of claim 1, wherein, in the step (2), the cellulose is employed in an amount of 2 to 5 g.

4. The method of claim 1, wherein, in the step (3), a molar ratio of sodium periodate to the regenerated cellulose aerogel is in a range of 1 to 1.5, and the deionized water is employed in an amount of 150 to 500 mL.

5. The method of claim 1, wherein, in the step (4), the dialdehyde cellulose aerogel, anhydrous ethanol, polyethyleneimine, and sodium cyanoborohydride are employed in an amount of 1 to 1.5 g, 50 to 200 mL, 2.7 to 4.5 g, and 3 to 5 g, respectively.

6. The method of claim 1, wherein, in the step (5), the 4-formylphenylboronic acid, anhydrous ethanol, PEI-modified cellulose aerogel, and sodium cyanoborohydride are employed in an amount of 3 to 5 g, 100 to 500 mL, 1 to 3 g, and 5 to 8 g, respectively.

Description

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

(1) The disclosure will now be described further by way of examples.

(2) In an embodiment, a method for preparing a modified cellulose aerogel for glycoprotein separation, comprising steps of:

(3) (1) adding 6 to 10 g of NaOH and 9 to 13 g of urea to a volume of 77 to 85 mL of deionized water and stirring to form a first solution, which is pre-cooled at −12.5° C.;

(4) (2) into the pre-cooled first solution, 2 to 5 g of cellulose (cotton linter pulp, DP=600, with α-cellulose content of above 95%) is added with vigorous stirring and completely dissolved and then a volume of 2 to 5 mL of ECH is added to form a second solution, which is poured into a mould and allowed to stand for 24 h and is then washed and freeze dried to obtain a regenerated cellulose aerogel (R-CA);

(5) (3) adding 1 mol of the R-CA and 1 to 1.5 mol of NaIO.sub.4 to a volume of 150 to 500 mL of deionized water and stirring to form a third solution, which is subjected to a reaction in the dark at a temperature of 50 to 70° C. for 5 to 7 h, at the end of which time a volume of 3 to 5 mL of ethylene glycol is added and stirred for 30 to 60 min followed by washing with deionized water and ethanol and then freeze drying to obtain a dialdehyde cellulose aerogel (D-CA);

(6) (4) adding 1 to 1.5 g of the D-CA to a volume of 50 to 200 mL of anhydrous ethanol to form a fourth solution, to which 2.7 to 4.5 g of PEI and 3 to 5 g of CH.sub.3BNNa are added to conduct a reaction at a temperature of 50 to 70° C. for 6 h followed by washing with a large amount of ethanol to neutral and then freeze drying to obtain a PEI-modified cellulose aerogel (PEI-CA); and

(7) (5) dissolving 3 to 5 g of 4-formylphenylboronic acid in a volume of 100 to 500 mL of anhydrous ethanol to form a fifth solution, to which 1 to 3 g of the PEI-CA and 5 to 8 g of NaCNBH.sub.3 are added to conduct a reaction at 50° C. for 12 to 24 h followed by washing with distilled water and ethanol and then vacuum drying to obtain a PEI/PBA-modified cellulose aerogel (PEI-PBA-CA).

Example 1

(8) 6 g of NaOH and 9 g of urea were added to 85 mL of deionized water and well stirred to form a first solution. The first solution was then pre-cooled at −12.5° C.

(9) 2 g of cotton linter pulp was added to the pre-cooled first solution with vigorous stirring and completely dissolved therein. Thereto was added 2 mL of ECH to form a uniform second solution by stirring. The second solution was poured into a mould and allowed to stand for 24 h, followed by washing and freeze drying to obtain R-CA.

(10) 1 mol of the R-CA and 1 mol of NaIO.sub.4 were added to 150 mL of deionized water and well stirred to form a third solution. The third solution was subjected to a reaction in the dark at 50° C. for 5 h. At the end of the reaction, 3 mL of ethylene glycol was added to the reaction mixture and stirred for 30 min. The resulting reaction product was washed with deionized water and ethanol and then freeze dried to obtain D-CA.

(11) 1 g of the D-CA was added to 50 mL of anhydrous ethanol to form a fourth solution. 2.7 g of PEI and 3 g of CH.sub.3BNNa were added thereto to conduct a reaction at 50° C. for 6 h. The reaction product was washed with a large amount of ethanol to neutral and then freeze dried to obtain PEI-CA.

(12) 3 g of 4-formylphenylboronic acid was dissolved in 100 mL of anhydrous ethanol to form a fifth solution. 1 g of the PEI-CA and 5 g of NaCNBH.sub.3 were added thereto to conduct a reaction at 50° C. for 12 h. The reaction product was washed with distilled water and ethanol and then vacuum dried to obtain PEI/PBA-CA.

Example 2

(13) 7 g of NaOH and 12 g of urea were added to 81 mL of deionized water and well stirred to form a first solution. The first solution was then pre-cooled at −12.5° C.

(14) 3 g of cotton linter pulp was added to the pre-cooled first solution with vigorous stirring and completely dissolved therein. Thereto was added 2 mL of ECH to form a uniform second solution by stirring. The second solution was poured into a mould and allowed to stand for 24 h, followed by washing and freeze drying to obtain R-CA.

(15) 1 mol of the R-CA and 1.1 mol of NaIO.sub.4 were added to 200 mL of deionized water and well stirred to form a third solution. The third solution was subjected to a reaction in the dark at 70° C. for 5 h. At the end of the reaction, 4 mL of ethylene glycol was added to the reaction mixture and stirred for 60 min. The resulting reaction product was washed with deionized water and ethanol and then freeze dried to obtain D-CA.

(16) 1.5 g of the D-CA was added to 150 mL of anhydrous ethanol to form a fourth solution. 4.0 g of PEI and 5 g of CH.sub.3BNNa were added thereto to conduct a reaction at 70° C. for 6 h. The reaction product was washed with a large amount of ethanol to neutral and then freeze dried to obtain PEI-CA.

(17) 3 g of 4-formylphenylboronic acid was dissolved in 300 mL of anhydrous ethanol to form a fifth solution. 3 g of the PEI-CA and 8 g of NaCNBH.sub.3 were added thereto to conduct a reaction at 50° C. for 24 h. The reaction product was washed with distilled water and ethanol and then vacuum dried to obtain PEI/PBA-CA.

Example 3

(18) 10 g of NaOH and 9 g of urea were added to 81 mL of deionized water and well stirred to form a first solution. The first solution was then pre-cooled at −12.5° C.

(19) 4 g of cotton linter pulp was added to the pre-cooled first solution with vigorous stirring and completely dissolved therein. Thereto was added 5 mL of ECH to form a uniform second solution by stirring. The second solution was poured into a mould and allowed to stand for 24 h, followed by washing and freeze drying to obtain R-CA.

(20) 1 mol of the R-CA and 1.5 mol of NaIO.sub.4 were added to 300 mL of deionized water and well stirred to form a third solution. The third solution was subjected to a reaction in the dark at 70° C. for 7 h. At the end of the reaction, 5 mL of ethylene glycol was added to the reaction mixture and stirred for 60 min. The resulting reaction product was washed with deionized water and ethanol and then freeze dried to obtain D-CA.

(21) 1.5 g of the D-CA was added to 200 mL of anhydrous ethanol to form a fourth solution. 2.7 g of PEI and 4.5 g of CH.sub.3BNNa were added thereto to conduct a reaction at 50° C. for 6 h. The reaction product was washed with a large amount of ethanol to neutral and then freeze dried to obtain PEI-CA.

(22) 4 g of 4-formylphenylboronic acid was dissolved in 450 mL of anhydrous ethanol to form a fifth solution. 2.5 g of the PEI-CA and 6 g of NaCNBH.sub.3 were added thereto to conduct a reaction at 50° C. for 12 h. The reaction product was washed with distilled water and ethanol and then vacuum dried to obtain PEI/PBA-CA.

Example 4

(23) 10 g of NaOH and 10 g of urea were added to 80 mL of deionized water and well stirred to form a first solution. The first solution was then pre-cooled at −12.5° C.

(24) 5 g of cotton linter pulp was added to the pre-cooled first solution with vigorous stirring and completely dissolved therein. Thereto was added 5 mL of ECH to form a uniform second solution by stirring. The second solution was poured into a mould and allowed to stand for 24 h, followed by washing and freeze drying to obtain R-CA.

(25) 1 mol of the R-CA and 1.3 mol of NaIO.sub.4 were added to 500 mL of deionized water and well stirred to form a third solution. The third solution was subjected to a reaction in the dark at 70° C. for 7 h. At the end of the reaction, 5 mL of ethylene glycol was added to the reaction mixture and stirred for 30 min. The resulting reaction product was washed with deionized water and ethanol and then freeze dried to obtain D-CA.

(26) 1.2 g of the D-CA was added to 100 mL of anhydrous ethanol to form a fourth solution. 3.5 g of PEI and 3.5 g of CH.sub.3BNNa were added thereto to conduct a reaction at 70° C. for 6 h. The reaction product was washed with a large amount of ethanol to neutral and then freeze dried to obtain PEI-CA.

(27) 4.5 g of 4-formylphenylboronic acid was dissolved in 100 to 500 mL of anhydrous ethanol to form a fifth solution. 3 g of the PEI-CA and 7.5 g of NaCNBH.sub.3 were added thereto to conduct a reaction at 50° C. for 24 h. The reaction product was washed with distilled water and ethanol and then vacuum dried to obtain PEI/PBA-CA.

Example 5

(28) 6 g of NaOH and 13 g of urea were added to 81 mL of deionized water and well stirred to form a first solution. The first solution was then pre-cooled at −12.5° C.

(29) 4 g of cotton linter pulp was added to the pre-cooled first solution with vigorous stirring and completely dissolved therein. Thereto was added 3.5 mL of ECH to form a uniform second solution by stirring. The second solution was then poured into a mould and allowed to stand for 24 h, followed by washing and freeze drying to obtain R-CA.

(30) 1 mol of the R-CA and 1.1 mol of NaIO.sub.4 were added to 350 mL of deionized water and well stirred to form a third solution. The third solution was subjected to a reaction in the dark at 65° C. for 7 h. At the end of the reaction, 5 mL of ethylene glycol was added to the reaction mixture and stirred for 60 min. The resulting reaction product was washed with deionized water and ethanol and then freeze dried to obtain D-CA.

(31) 1 g of the D-CA was added to 50 to 200 mL of anhydrous ethanol to form a fourth solution. 4.5 g of PEI and 5 g of CH.sub.3BNNa were added thereto to conduct a reaction at 70° C. for 6 h. The reaction product was washed with a large amount of ethanol to neutral and then freeze dried to obtain PEI-CA.

(32) 5 g of 4-formylphenylboronic acid was dissolved in 500 mL of anhydrous ethanol to form a fifth solution. 2.5 g of the PEI-CA and 6 g of NaCNBH.sub.3 were added thereto to conduct a reaction at 50° C. for 24 h. The reaction product was washed with distilled water and ethanol and then vacuum dried to obtain PEI/PBA-CA.

(33) The method of the disclosure provides at least the following advantages.

(34) First, the method is simple and environmentally safe, and the materials used by this method have good biocompatibility.

(35) Next, the use of PEI to modify the cellulose aerogel substrate can improve the selectivity and identification capability of the phenylboronic acid to be bound to the substrate for glycoproteins, and thus improve its separation capability of separating the glycoproteins from the complex biological samples or clinical samples.

(36) Further, with the PEI/PBA-CA prepared by the present method, it is possible to achieve simple and effective separation of glycoproteins. In the glycoprotein separation using the PEI/PBA-CA, as mentioned in the background, when the pH of the surrounding solution is basic, the boronic acid presents as the form of a tetragonal boronate anion (sp.sup.3), which can be esterified with the cis-diol groups of the glycoproteins to form covalent bonds therebetween; while when the surrounding pH becomes acidic, the boronic acid presents as a trigonal configuration (sp.sup.2), resulting in dissociation of the ester.