CROSSLINKED PROTEIN-BASED SEPARATION MEMBRANE AND APPLICATION THEREOF

Abstract

A crosslinked protein-based separation membrane and application thereof. The separation membrane is formed by attaching a crosslinked protein nanomembrane to a porous membrane, the crosslinked protein nanomembrane is formed by crosslinking a two-dimensional nanomembrane which is formed by phase transition of a protein with a crosslinking agent, the separation membrane contains a dense surface layer and a support layer, the dense surface layer is the crosslinked protein nanomembrane, and the support layer is the porous membrane; the protein is any one of lysozyme, bovine serum albumin, insulin, and α-lactalbumin; the crosslinked protein-based separation membrane has a good biocompability, may serve as a dialysis membrane for blood purification, and has a higher retention ratio for large molecular proteins.

Claims

1-14. (canceled)

15. A crosslinked lysozyme-based dialysis membrane, wherein the dialysis membrane is formed by attaching a crosslinked lysozyme nanomembrane to a PET nuclear track membrane, the crosslinked lysozyme nanomembrane is formed by crosslinking a two-dimensional nanomembrane which is formed by phase transition of a lysozyme with a glutaraldehyde, the dialysis membrane contains a dense surface layer and a support layer, the dense surface layer is the crosslinked lysozyme nanomembrane, and the support layer is the PET nuclear track membrane; a method of forming a lysozyme two-dimensional nanomembrane by phase transition of the lysozyme comprises: adjusting a pH value of a 10-100 mmol/L 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of tris(2-carboxyethyl) phosphine to 6.0-8.0 by using NaOH to get an adjusted solution, then mixing the adjusted solution with a 1-30 mg/mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of the lysozyme in an isovolumetric manner to get a mixed solution, spreading the mixed solution to a whole surface of a substrate directly, incubating at room temperature for 2-6 hours, forming a layer of the lysozyme two-dimensional nanomembrane on a gas-liquid interface of a liquid on the substrate; a method of crosslinking the lysozyme two-dimensional nanomembrane which is formed by phase transition of the lysozyme with the glutaraldehyde comprises: transferring the lysozyme two-dimensional nanomembrane to a glutaraldehyde aqueous solution with a mass fraction of 0.2%-2% for crosslinking for 2-6 hours at room temperature.

16. The crosslinked lysozyme-based dialysis membrane according to claim 15, wherein a diameter of the PET nuclear track membrane is 25 mm, a thickness of the PET nuclear track membrane is 12 μm, a pore size of the PET nuclear track membrane is in a range of 1-10 μm.

17. Use of the crosslinked lysozyme-based dialysis membrane in the separation of mixed proteins according to claim 15, wherein the mixed proteins are bovine serum albumin and insulin, or muscle hemoglobin and insulin.

18. Use of the crosslinked lysozyme-based dialysis membrane in the separation of mixed dyes according to claim 15, wherein the mixed dyes are methyl blue and methyl orange, or methyl blue and Rhodamine B.

19. Use of the crosslinked lysozyme-based dialysis membrane in the removal of urotoxin according to claim 15, wherein the urotoxin is any one of urea, creatinine, β-microglobulin and indoxyl sulfate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is a diagram of a scanning electron micrograph of a crosslinked lysozyme nanomembrane in Embodiment 1.

[0016] FIG. 2 is a planar diagram of a transmission electron microscope (TEM) of the crosslinked lysozyme nanomembrane in Embodiment 1.

[0017] FIG. 3 is a diagram of an atomic force microscope (AFM) of the crosslinked lysozyme nanomembrane in Embodiment 1.

[0018] FIG. 4 shows pore size distribution of crosslinked lysozyme nanomembranes with different thicknesses.

[0019] FIG. 5 shows the removal of urea, indoxyl sulfate, creatinine and β-microglobulin by a dialysis membrane obtained from Embodiment 1.

DETAILED DESCRIPTION OF THE INVENTION

The Best Embodiment of the Invention

Embodiment 1

[0020] A pH value of a 60 μL 50 mmol/L 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of tris(2-carboxyethyl) phosphine was adjusted to 7.0 by using NaOH; and then, the adjusted solution was mixed, in an isovolumetric manner, with a 60 μL 2 mg/mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of lysozyme evenly before directly spreading a resulting mixture to a whole surface of a 18 mm×18 mm cover glass; and the mixed solution was incubated at room temperature for 2 hours to form a layer of lysozyme two-dimensional nanomembrane on a gas-liquid interface of a liquid on the cover glass; the lysozyme two-dimensional nanomembrane on the gas-liquid interface was transferred to an glutaraldehyde aqueous solution having a mass fraction of 1%, and then crosslinked for 2 hours at room temperature to obtain a crosslinked lysozyme nanomembrane.

[0021] The above crosslinked lysozyme nanomembrane was attached on a PET nuclear track membrane with a diameter of 25 mm, a thickness of 12 μm and a pore size of 10 μm to obtain a dialysis membrane with the crosslinked lysozyme nanomembrane as a dense surface layer and the PET nuclear track membrane as a support layer.

The Embodiments of the Invention

Embodiment 2

[0022] In this embodiment, 2 mg/mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of lysozyme in Embodiment 1 was replaced with an isovolumetric 4 mg/mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of lysozyme; and other steps were the same as those of Embodiment 1 to obtain a dialysis membrane.

Embodiment 3

[0023] In this embodiment, 2 mg/mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of lysozyme in Embodiment 1 was replaced with an isovolumetric 6 mg/mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of lysozyme; and other steps were the same as those of Embodiment 1 to obtain a dialysis membrane.

Embodiment 4

[0024] In this embodiment, 2 mg/mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of lysozyme in Embodiment 1 was replaced with an isovolumetric 8 mg/mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of lysozyme; and other steps were the same as those of Embodiment 1 to obtain a dialysis membrane.

Embodiment 5

[0025] In this embodiment, 2 mg/mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of lysozyme in Embodiment 1 was replaced with an isovolumetric 10 mg/mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of lysozyme; and other steps were the same as those of Embodiment 1 to obtain a dialysis membrane.

Embodiment 6

[0026] In this embodiment, 2 mg/mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of lysozyme in Embodiment 1 was replaced with an isovolumetric 20 mg/mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of lysozyme; and other steps were the same as those of Embodiment 1 to obtain a dialysis membrane.

Embodiment 7

[0027] In this embodiment, 2 mg/mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of lysozyme in Embodiment 1 was replaced with an isovolumetric 30 mg/mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of lysozyme; and other steps were the same as those of Embodiment 1 to obtain a dialysis membrane.

[0028] The inventor performed SEM characterization on the crosslinked lysozyme nanomembranes prepared in Embodiments 1-7; and the results showed that the thicknesses of the corresponding films were successively 50 nm (see FIG. 1), 60 nm, 90 nm, 100 nm, 120 nm, 220 nm and 250 nm; and the nanomembranes were densely-piled by 20 nm oligomers after phase transition of lysozyme (as shown in FIG. 2 and FIG. 3). The area of the prepared crosslinked lysozyme nanomembrane was controlled by a PET with a pore size of 6 mm; the membrane was made to float on 5 mL ultrapure water; and then 50 μL 0.5 mg/mL PEG with different molecular weights was dropwisely added on the membrane; then an UV-Vis absorption spectrum was used to monitor the permeability of the solution standing for around 24 hours, and to analyze the pore size distribution of the membrane; and the results were shown in FIG. 4. The pore size distribution of the membrane was obtained by fitting the retention ratios of the membrane for different molecular weights; and the pore size of the membrane decreased to 1.8 nm from 3.4 nm with the increase of the membrane thickness.

Embodiment 8

[0029] Use of the dialysis membrane of Embodiment 1 in the separation of methyl blue and methyl orange, methyl blue and Rhodamine B, muscle hemoglobin and insulin, bovine serum albumin and insulin was specifically as follows:

a dialysis membrane was made to float on 5 mL ultrapure water; and the membrane was separately dropwisely added with a 50 μL aqueous solution containing 50 mg/L methyl blue and 50 mg/L methyl orange, a 50 μL aqueous solution containing 50 mg/L methyl blue and 50 mg/L Rhodamine, a 50 μL aqueous solution containing 10 g/L muscle hemoglobin and 10 g/L insulin, and a 50 μL aqueous solution containing 10 g/L bovine serum albumin and 10 g/L insulin; after the solution stood for 24 hours at room temperature, the permeability of the solution was monitored by an UV Vis absorption spectrum. The experimental results show that the dialysis membrane can completely pass through molecules having a molecular diameter of less than 2 nm; moreover, the methyl blue, bovine serum albumin and muscle hemoglobin having a molecular diameter of more than 3 nm and carrying negative charges can be completely intercepted; the retention ratio of methyl orange is merely 0.3%, the retention ratio of Rhodamine B is 1.4%, and the retention ratio of insulin is 22.6%. The diffusion rate of methyl orange is up to 606 nmol cm.sup.−2 h.sup.−1, and the diffusion rate of Rhodamine B is 308 nmol cm.sup.−2 h.sup.−1. Therefore, the membrane can achieve the rapid and effective separation from methyl blue and methyl orange, methyl blue and Rhodamine, muscle hemoglobin and insulin, bovine serum albumin and insulin.

Embodiment 9

[0030] Use of the dialysis membrane of Embodiment 1 in the removal of urea, creatinine, β-microglobulin and indoxyl sulfate was specifically as follows:

the simulation solution was an aqueous solution containing 1 mg/mL bovine serum albumin, 25 mg/L indoxyl sulfate, 40 mg/L β-microglobulin, 100 mg/L creatinine and 1.5 mg/mL urea; flow velocity of the simulation solution and dialysis fluid (water) was 10 mL/min; and dialysis was conducted for 4 hours with a dialysis membrane. The removal ratio of the dialysis membrane for bovine serum albumin, indoxyl sulfate, β-microglobulin, creatinine and urea was tested. As shown in FIG. 5, the dialysis membrane can intercept the macromolecular bovine serum albumin; and has better removal effects for indoxyl sulfate, urea, β-microglobulin, and creatinine; and the removal ratios were respectively 33.1%, 82.2%, 50.3% and 81.3%.

Embodiment 10

[0031] A pH value of a 60 μL 50 mmol/L 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of tris(2-carboxyethyl) phosphine was adjusted to 5.0 by using NaOH; and then, the adjusted solution was mixed with a 60 μL 5 mg/mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of bovine serum albumin evenly before directly spreading a resulting mixture to a whole surface of a 18 mm×18 mm cover glass; and the mixed solution was incubated at room temperature for 2 hours to form a layer of bovine serum albumin two-dimensional nanomembrane on a gas-liquid interface of a liquid on the cover glass; the bovine serum albumin two-dimensional nanomembrane on the gas-liquid interface was transferred to a Genipin aqueous solution having a mass fraction of 1%, and then crosslinked for 2 hours at room temperature to obtain a crosslinked bovine serum albumin nanomembrane. The obtained crosslinked bovine serum albumin nanomembrane was attached on a PC membrane with a diameter of 25 mm, a thickness of 12 μm and a pore size of 10 μm to obtain a dialysis membrane with the crosslinked bovine serum albumin nanomembrane as a dense surface layer and the PC membrane as a support layer. The use of the dialysis membrane in the removal of urea, creatinine, β-microglobulin and indoxyl sulfate was the same as that in Embodiment 9. The experimental results show that the dialysis membrane can intercept the macromolecular bovine serum albumin; and has better removal effects for indoxyl sulfate, urea, β-microglobulin, and creatinine; and the removal ratios were respectively 44.2%, 90.2%, 60.4% and 88.6%.

Embodiment 11

[0032] A pH value of a 60 μL 50 mmol/L 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of tris(2-carboxyethyl) phosphine was adjusted to 4.0 by using NaOH; and then, the adjusted solution was mixed with a 60 μL 2 mg/mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of α-lactalbumin evenly before directly spreading a resulting mixture to a whole surface of a 18 mm×18 mm cover glass; and the mixed solution was incubated at room temperature for 2 hours to form a layer of α-lactalbumin two-dimensional nanomembrane on a gas-liquid interface of a liquid on the cover glass; the α-lactalbumin two-dimensional nanomembrane on the gas-liquid interface was transferred to a glutamine transaminase aqueous solution having a mass fraction of 1%, and then crosslinked for 2 hours at room temperature to obtain a crosslinked α-lactalbumin nanomembrane. The obtained crosslinked α-lactalbumin nanomembrane was attached on a PP membrane with a diameter of 25 mm, a thickness of 12 μm and a pore size of 10 μm to obtain a dialysis membrane with the crosslinked α-lactalbumin nanomembrane as a dense surface layer and the PP membrane as a support layer. The use of the dialysis membrane in the removal of urea, creatinine, β-microglobulin and indoxyl sulfate was the same as that in Embodiment 9. The experimental results show that the use of the dialysis membrane in the removal of urea, creatinine, β-microglobulin and indoxyl sulfate was the same as that in Embodiment 9; and the dialysis membrane can completely intercept the macromolecular bovine serum albumin; and has better removal effects for indoxyl sulfate, urea, β-microglobulin, and creatinine; and the removal ratios were respectively 35.1%, 89.2%, 51.4% and 87.5%.

Embodiment 12

[0033] A pH value of a 60 μL 50 mmol/L 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of tris(2-carboxyethyl) phosphine was adjusted to 6.0 by using NaOH; and then, the adjusted solution was mixed with a 60 μL 2 mg/mL 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffered solution of insulin evenly before directly spreading a resulting mixture to a whole surface of a 18 mm×18 mm cover glass; and the mixed solution was incubated at room temperature for 2 hours to form a layer of insulin two-dimensional nanomembrane on a gas-liquid interface of a liquid on the cover glass; the insulin two-dimensional nanomembrane on the gas-liquid interface was transferred to a 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride aqueous solution having a mass fraction of 1%, and then crosslinked for 2 hours at room temperature to obtain a crosslinked insulin nanomembrane. The obtained crosslinked insulin nanomembrane was attached on a PSF membrane with a diameter of 25 mm, a thickness of 12 μm and a pore size of 10 μm to obtain a dialysis membrane with the crosslinked insulin nanomembrane as a dense surface layer and the PSF membrane as a support layer. The use of the dialysis membrane in the removal of urea, creatinine, β-microglobulin and indoxyl sulfate was the same as that in Embodiment 9. The experimental results show that the use of the dialysis membrane in the removal of urea, creatinine, β-microglobulin and indoxyl sulfate was the same as that in Embodiment 9; and the dialysis membrane can completely intercept the macromolecular bovine serum albumin; and has better removal effects for indoxyl sulfate, urea, β-microglobulin, and creatinine; and the removal ratios were respectively 32.1%, 83.6%, 50.1% and 86.1%.