TWI433716B - A nanofiber membrane, a production process and use thereof - Google Patents

A nanofiber membrane, a production process and use thereof Download PDF

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TWI433716B
TWI433716B TW100122738A TW100122738A TWI433716B TW I433716 B TWI433716 B TW I433716B TW 100122738 A TW100122738 A TW 100122738A TW 100122738 A TW100122738 A TW 100122738A TW I433716 B TWI433716 B TW I433716B
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nanofiber membrane
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chitosan
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membrane
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TW201300164A (en
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Tai Hong Cheng
Cheng Chiang Huang
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Taiwan Textile Res Inst
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Description

奈米纖維膜、其製造方法及用途Nanofiber membrane, manufacturing method and use thereof

本發明係關於一種奈米纖維膜，其具有一胺基，該胺基係視情況經反應性染料、縮水甘油三甲基氯化銨(GTAC)或溴乙酸(bromoacetic acid)改質；其製造方法以及其用途。The present invention relates to a nanofiber membrane having an amine group which is optionally modified with a reactive dye, glycidyltrimethylammonium chloride (GTAC) or bromoacetic acid; Method and its use.

現今，為了獲得高純度的化學產物、提高化學製程中所欲化合物或產物之轉化率以及降低製造成本，分離技術廣泛地用於各種化學製程中，並且持續地改良中。Today, in order to obtain high purity chemical products, increase the conversion rate of desired compounds or products in chemical processes, and reduce manufacturing costs, separation techniques are widely used in various chemical processes and are continuously being improved.

一般而言，習知的分離方法係包括膠體過濾法、離子交換法以及親和層析法，其中該膠體過濾法係藉由欲分離物質之形狀或大小而分離；該離子交換法係藉由欲分離物質之電荷而分離；以及該親和層析法係藉由欲分離物質之特異性鍵結而分離。In general, conventional separation methods include a colloidal filtration method, an ion exchange method, and an affinity chromatography method in which the colloidal filtration method is separated by the shape or size of the substance to be separated; The charge of the separated material is separated; and the affinity chromatography is separated by specific bonding of the substance to be separated.

就離子交換法而言，分離過程中所使用之載體種類主要包括粒狀樹脂、層狀薄膜及奈米纖維膜。不同的載體具有不同的分離特性及要求。例如：對於常見的填充床層析法中所使用之粒狀樹脂而言，該樹脂必須要具有大小一致、材質堅固及耐高壓等特性；而就填充該樹脂之管柱而言，若粒狀樹脂堆疊緊密，亦即降低顆粒間之空間，則降低產生亂流的機會，以及若顆粒尺寸降低，雖然會降低欲分離之流體的流率，但此可降低亂流空間，並增加分離效果。In the case of the ion exchange method, the types of carriers used in the separation process mainly include a granular resin, a layered film, and a nanofiber film. Different carriers have different separation characteristics and requirements. For example, for the granular resin used in the common packed bed chromatography, the resin must have the characteristics of uniform size, firm material and high pressure resistance; and for the column filled with the resin, if it is granular The tight stacking of the resin, that is, reducing the space between the particles, reduces the chance of turbulence, and if the particle size is reduced, although the flow rate of the fluid to be separated is lowered, this reduces the turbulent space and increases the separation effect.

就分離特性而言，離子交換樹脂一般可分為強酸型陽離子交換樹脂、弱酸型陽離子交換樹脂、強鹼型陰離子交換樹脂以及弱鹼型陰離子交換樹脂。In terms of separation characteristics, ion exchange resins are generally classified into a strong acid type cation exchange resin, a weak acid type cation exchange resin, a strong base type anion exchange resin, and a weak base type anion exchange resin.

此外，就分離或純化蛋白質之應用而言，典型的分離或純化方法係包括藉由凝膠層析法、使用陰陽離子吸附薄膜以及高特異性奈米纖維膜，其中該奈米纖維膜特別有利於分離或純化程序，且因其具有高便利性、相較於傳統粒狀膠體樹脂，具有高結構穩定性、高孔隙度、高比表面積且無阻塞問題、高再生性、低壓降及高流量等特性而被受矚目。In addition, in the application for separating or purifying proteins, typical separation or purification methods include gel chromatography, use of an anion-cation adsorption film, and a high-specificity nanofiber membrane, wherein the nanofiber membrane is particularly advantageous. For separation or purification procedures, and because of its high convenience, compared to traditional granular colloidal resin, it has high structural stability, high porosity, high specific surface area and no blockage problem, high regenerability, low pressure drop and high flow rate. I was noticed by other characteristics.

US 2004/0241436A1揭露一種含有一聚合物和一蛋白質之奈米纖維，其中該蛋白質和該聚合物係藉由靜電紡絲(electrospinning)法形成一蛋白質薄膜組成物，而蛋白質係以共價地鍵結至纖維。US 2004/0241436 A1 discloses a nanofiber comprising a polymer and a protein, wherein the protein and the polymer form a protein film composition by electrospinning, and the protein is covalently bonded. Knot to fiber.

然而，目前仍有相較於上述專利文獻以及先前技術中已知之薄膜特性更佳的分離薄膜及使用該薄膜的分離方法之需求，以增強分離效果和降低製造標的產物成本。However, there is still a need for a separation film which is superior in film properties as known from the above-mentioned patent documents and the prior art, and a separation method using the same, to enhance the separation effect and to reduce the cost of manufacturing the target product.

因此，本發明之一目的係提供一新穎的具有胺基之奈米纖維膜，以及進一步提供一新穎的陰陽離子交換奈米纖維膜、一新穎的雙極性複合型奈米纖維膜、一新穎的固定化金屬螯合複合型奈米纖維膜與一新穎的染料親和性複合型奈米纖維膜。Accordingly, it is an object of the present invention to provide a novel nanofiber membrane having an amine group, and further to provide a novel anion-cation exchange nanofiber membrane, a novel bipolar composite nanofiber membrane, and a novel one. The immobilized metal chelate composite nanofiber membrane is combined with a novel dye affinity composite nanofiber membrane.

本發明之另一目的係提供上述薄膜之新穎製造方法及其用於廢水處理(例如處理廢水中之重金屬和有機物質)、過濾細菌或蛋白質純化之用途。Another object of the present invention is to provide a novel process for the manufacture of the above-described films and their use for wastewater treatment (e.g., treatment of heavy metals and organic materials in wastewater), filtration of bacteria or protein purification.

本發明之特點可參閱本案圖式及以下較佳實施方式之詳細說明而獲得清楚地瞭解。The features of the present invention can be clearly understood from the description of the drawings and the detailed description of the preferred embodiments.

本文中使用的術語“一”和“該”是同義詞的並且可以與“一種或多種”和“至少一種”可互換使用，除非該語言和/或上下文另外清楚指明。相應地，例如，在這裏或在所附申請專利範圍中對於“具有氰基之聚合物”的提及能夠指單種具有氰基之聚合物或一種以上的具有氰基之聚合物。另外，全部數值，除非另外具體地指出，被理解為被詞“約”修飾。The terms "a", "an" and "the" are used interchangeably, and may be used interchangeably with "one or more" and "at least one" unless the language and/or context clearly dictates otherwise. Accordingly, reference to "a polymer having a cyano group" herein or in the scope of the appended claims can refer to a single polymer having a cyano group or more than one polymer having a cyano group. In addition, all numerical values, unless specifically stated otherwise, are understood to be modified by the word "about."

在一個具體實施例中，本發明提供一種具有胺基之奈米纖維膜，其中該胺基視情況可經3-縮水甘油氧基丙基-三甲氧基矽烷(GPTMS)、反應性染料、縮水甘油三甲基氯化銨(GTAC)或溴乙酸(bromoacetic acid)改質。In a specific embodiment, the present invention provides a nanofiber membrane having an amine group, wherein the amine group may optionally pass through 3-glycidoxypropyl-trimethoxydecane (GPTMS), a reactive dye, and shrinkage. Trimethylammonium chloride (GTAC) or bromoacetic acid is modified.

在一個具體實施例中，本發明所提供之具有胺基之奈米纖維膜係由一具有胺基之聚合物所構成。In a specific embodiment, the nanofiber membrane having an amine group provided by the present invention is composed of a polymer having an amine group.

在另一個具體實施例中，本發明所提供之具有胺基之奈米纖維膜係藉由靜電紡絲方法將具有氰基之聚合物紡絲，並進行官能基改質而獲得。In another embodiment, the nanofiber membrane having an amine group provided by the present invention is obtained by spinning a polymer having a cyano group by an electrospinning method and modifying a functional group.

在另一個具體實施例中，本發明之具有胺基之奈米纖維膜可利用3-縮水甘油氧基丙基-三甲氧基矽烷(GPTMS)、反應性染料、縮水甘油三甲基氯化銨(GTAC)或溴乙酸對於該胺基作進一步地改質，其中反應性染料包含Cibacron Blue F3GA、Procion red HE3B、Green A dye、Blue Dextran，較佳為Cibacron Blue F3GA。In another embodiment, the aminocellulose membrane having an amine group of the present invention can utilize 3-glycidoxypropyl-trimethoxydecane (GPTMS), a reactive dye, and glycidyl trimethylammonium chloride. The amine group is further modified by (GTAC) or bromoacetic acid, wherein the reactive dye comprises Cibacron Blue F3GA, Procion red HE3B, Green A dye, Blue Dextran, preferably Cibacron Blue F3GA.

Cibacron Blue F3GA是一種多芳香環的磺化物，其具有式(1)結構：Cibacron Blue F3GA is a polyaromatic sulfonate having the structure of formula (1):

這種藍色染料由於它具有與NAD+ 相似的空間結構，所以它與各種激酶、脫氫酶、血清蛋白、溶菌酶、DNA聚合酶等具有特異性，常用於親和性層析分離。Cibacron Blue F3GA的三氮六環中的氯與反應基團能夠與胺基(-NH2 )反應。This blue dye has specificity to various kinases, dehydrogenases, serum proteins, lysozyme, DNA polymerase, etc., and is often used for affinity chromatography separation because it has a similar spatial structure to NAD + . The chlorine and reactive groups in the triazine ring of Cibacron Blue F3GA are capable of reacting with the amine group (-NH 2 ).

在另一個具體實例中，一種製造上述該等多功能性奈米纖維膜之方法，其包含下列步驟：In another embodiment, a method of making the above-described multifunctional nanofiber membranes comprising the steps of:

(1)將一具有氰基之奈米纖維膜直接與二胺化合物於30至95℃溫度，較佳80至95℃下反應0.5至5小時，較佳2.5至3..5小時反應；或(1) reacting a nanofiber membrane having a cyano group directly with a diamine compound at a temperature of 30 to 95 ° C, preferably 80 to 95 ° C, for 0.5 to 5 hours, preferably 2.5 to 3. 5 hours; or

(2)將該奈米纖維膜之氰基以鹼化水解處理部分地或全部地改質為羧基；將該具有羧基之奈米纖維膜與氯化硫醯基於30至90℃，較佳50至70℃溫度下反應0.1至3小時，較佳0.5至1小時，以改質羧基為醯氯基；以及將該具有醯氯基之奈米纖維膜與該二胺化合物於30至95℃溫度，較佳80至95℃下反應0.5至5小時，較佳0.5至1小時；或(2) partially or completely modifying the cyano group of the nanofiber membrane to a carboxyl group by alkali hydrolysis treatment; and based on 30 to 90 ° C, preferably 50, of the nanofiber membrane having a carboxyl group; The reaction is carried out at a temperature of 70 ° C for 0.1 to 3 hours, preferably 0.5 to 1 hour, to modify the carboxyl group to be a fluorenyl chloride group; and the nanofiber membrane having a fluorenyl chloride group and the diamine compound are at a temperature of 30 to 95 ° C Preferably, the reaction is carried out at 80 to 95 ° C for 0.5 to 5 hours, preferably 0.5 to 1 hour; or

(3)將該奈米纖維膜之氰基以鹼化水解處理部分地或全部地改質為羧基後，再將該具有羧基之奈米纖維膜與幾丁聚醣於30至90℃溫度，較佳70至90℃下反應0.5至5小時，較佳2.5至3.5小時；或(3) after partially or completely modifying the cyano group of the nanofiber membrane to a carboxyl group by alkalization hydrolysis treatment, the nanofiber membrane having a carboxyl group and the chitosan at a temperature of 30 to 90 ° C, Preferably, the reaction is carried out at 70 to 90 ° C for 0.5 to 5 hours, preferably 2.5 to 3.5 hours; or

(4)將該奈米纖維膜之氰基以鹼化水解處理部分地或全部地改質為羧基；將該具有羧基之奈米纖維膜與氯化硫醯基於30至90℃，較佳50至70℃溫度下反應0.1至3小時，較佳0.5至1小時，以改質羧基為醯氯基；以及將該具有醯氯基之奈米纖維膜與幾丁聚醣於30至90℃溫度，較佳70至90℃下反應0.5至5小時，較佳2.5至3.5小時；以及(4) partially or completely modifying the cyano group of the nanofiber membrane to a carboxyl group by alkali hydrolysis treatment; and based on 30 to 90 ° C, preferably 50, of the nanofiber membrane having a carboxyl group; The reaction is carried out at a temperature of 70 ° C for 0.1 to 3 hours, preferably 0.5 to 1 hour, to modify the carboxyl group to be a fluorenyl chloride group; and the nanofiber membrane having a fluorenyl chloride group and the chitosan at a temperature of 30 to 90 ° C Preferably, the reaction is carried out at 70 to 90 ° C for 0.5 to 5 hours, preferably 2.5 to 3.5 hours;

視情況Subject to availability

(5)將步驟(1)至(4)中所得任一具有胺基之奈米纖維膜與一反應性染料、GTAC或溴乙酸於25至100℃，較佳50至70℃溫度下反應0.1至24小時，較佳1至5小時，(5) reacting any of the nanofiber membranes having an amine group obtained in the steps (1) to (4) with a reactive dye, GTAC or bromoacetic acid at a temperature of 25 to 100 ° C, preferably 50 to 70 ° C. Up to 24 hours, preferably 1 to 5 hours,

其中該具有氰基之奈米纖維膜係由具有氰基之聚合物所構成，該聚合物較佳為聚丙烯腈(PAN)或其共聚物。The nanofiber membrane having a cyano group is composed of a polymer having a cyano group, and the polymer is preferably polyacrylonitrile (PAN) or a copolymer thereof.

本發明方法中合適之二胺化合物包含乙二胺(EDA)、己二胺(HDA)、苯二胺、二胺基甲基環己烷、雙(胺甲基)吡啶，且較佳為乙二胺及己二胺。Suitable diamine compounds in the process of the invention comprise ethylenediamine (EDA), hexamethylenediamine (HDA), phenylenediamine, diaminomethylcyclohexane, bis(aminomethyl)pyridine, and preferably B. Diamine and hexamethylene diamine.

在本發明之一個具體實例中，鹼化水解處理係將具有氰基之奈米纖維膜於4至150℃、較佳60至90℃溫度下0.1至30 N、較佳1至5 N的鹼溶液中鹼化水解1至600分鐘、較 佳5至60分鐘後，以及視情況置於室溫下一0.01至10N、較佳0.1至1N的酸性溶液中1至1440分鐘、較佳120至720分鐘，其中該鹼溶液為NaOH溶液或KOH水溶液以及該酸性溶液為HCl水溶液。In a specific embodiment of the present invention, the alkali hydrolysis treatment is a base having a cyano nanofiber membrane at a temperature of 4 to 150 ° C, preferably 60 to 90 ° C, 0.1 to 30 N, preferably 1 to 5 N. hydrolyzing the solution basified with 1 to 600 minutes, compared with 60 minutes to 5, good, and optionally 0.01 to stand at room temperature next 10N, preferably 0.1 to 1N acid solution of 1 to 1,440 minutes, preferably from 120 to 720 Minutes, wherein the alkali solution is a NaOH solution or an aqueous KOH solution and the acidic solution is an aqueous HCl solution.

在本發明方法之一個具體實例中，反應性染料之濃度為0.05至10mg/ml、較佳0.1至1 mg/ml；GTAC之濃度為0.1至50M、較佳0.5至5 M；溴乙酸之濃度為0.001至1M、較佳0.01至0.05 M；二胺化合物之濃度為1至100%(體積/體積)、較佳50至100%(體積/體積)；氯化硫醯基之濃度為0.01至10 M、較佳0.3至3 M；以及幾丁聚醣之濃度為0.01至3%(重量/體積)、較佳0.1至1%(重量/體積)。In a specific embodiment of the method of the present invention, the concentration of the reactive dye is 0.05 to 10 mg/ml, preferably 0.1 to 1 mg/ml; the concentration of GTAC is 0.1 to 50 M, preferably 0.5 to 5 M; the concentration of bromoacetic acid It is 0.001 to 1 M, preferably 0.01 to 0.05 M; the concentration of the diamine compound is 1 to 100% (v/v), preferably 50 to 100% (vol/vol); the concentration of sulfonium chloride is 0.01 to 10 M, preferably 0.3 to 3 M; and the concentration of chitosan is 0.01 to 3% (weight/volume), preferably 0.1 to 1% (weight/volume).

在本發明方法之一個具體實例中，在步驟(3)之該幾丁聚醣與該奈米纖維膜之羧基反應以及步驟(4)之該幾丁聚醣與該奈米纖維膜之醯氯基反應中，可加入GPTMS且一起於25至100℃、較佳70至90℃溫度下反應0.1至24小時、較佳1至5小時，其中該幾丁聚醣之濃度為0.01至3%(重量/體積)、較佳0.1至1%(重量/體積)；該GPTMS之濃度為1至100%(體積/體積)、較佳10至50%(體積/體積)。所得經改質之奈米纖維膜具有式(2)官能基：In a specific embodiment of the method of the present invention, the chitosan of the step (3) reacts with a carboxyl group of the nanofiber membrane, and the chitosan of the step (4) and the chlorine of the nanofiber membrane In the base reaction, GPTMS may be added and reacted together at a temperature of 25 to 100 ° C, preferably 70 to 90 ° C for 0.1 to 24 hours, preferably 1 to 5 hours, wherein the concentration of the chitosan is 0.01 to 3% ( Weight/volume, preferably 0.1 to 1% (weight/volume); the concentration of the GPTMS is from 1 to 100% (vol/vol), preferably from 10 to 50% (vol/vol). The obtained modified nanofiber membrane has the functional group of formula (2):

本發明利用縮水甘油三甲基氯化銨(GTAC)修飾幾丁聚醣、乙二胺或己二胺的胺基端，使得所合成之產物可帶有四級胺基(例如式(3)、(4)及(5))，以增加正電荷基團的帶荷密度、進而提高PAN膜的離子性鍵結合力，並提供一強鹼型陰離子交換膜。The invention utilizes glycidyl trimethylammonium chloride (GTAC) to modify the amine end of chitosan, ethylenediamine or hexamethylenediamine, so that the synthesized product can carry a quaternary amine group (for example, formula (3) (4) and (5)) to increase the charge density of the positively charged group, thereby increasing the ionic bond bonding force of the PAN film, and providing a strong base type anion exchange membrane.

溴乙酸(BrA)是可將薄膜尾端所帶有之一級胺基轉變為兩個羧酸與二級胺基，並形成多齒配體狀態，可與金屬做螯合反應。因此，本發明提供一經溴乙酸改質之奈米纖維膜(其具有例如式(6)、(7)及(8)之官能基)，作為陽離子交換型奈米薄膜；和提供一經溴乙酸改質，接著再固定化金屬之奈米纖維膜，作為固定化金屬親和性薄膜。Bromoacetic acid (BrA) converts a primary amine group at the tail end of the film into two carboxylic acids and a secondary amine group, and forms a polydentate ligand state, which can chelate with the metal. Accordingly, the present invention provides a bromoacetic acid modified nanofiber membrane having functional groups such as formulas (6), (7) and (8) as a cation exchange type nanofilm; and providing a brominated acetic acid modification The metal nanofiber film is then immobilized as an immobilized metal affinity film.

本發明方法中具有氰基之奈米纖維膜可藉由熔噴法(Melt-blown)、模板聚合法(Template)、相分離法(Phase separation)、自組裝誘導法(Self-assembly)和靜電紡絲法等方法，較佳為靜電紡絲法，自具有氰基之聚合物如聚丙烯腈(PAN)或其共聚物製得。The nanofiber membrane having a cyano group in the method of the present invention can be melt-blown (Melt-blown), template polymerization, phase separation, self-assembly and self-assembly. A method such as a spinning method, preferably an electrospinning method, is produced from a polymer having a cyano group such as polyacrylonitrile (PAN) or a copolymer thereof.

本發明之奈米纖維膜的幾丁聚醣基團能夠與金屬離子產生螯合作用。此外，本發明之奈米纖維膜的反應性染料基團能夠對於金屬離子產生離子吸附作用。The chitosan group of the nanofiber membrane of the present invention is capable of chelation with metal ions. Further, the reactive dye group of the nanofiber membrane of the present invention is capable of generating ion adsorption for metal ions.

在一個具體實例中，本發明之奈米纖維膜可用於廢水處理、過濾細菌或蛋白質純化之用途，其中該廢水處理係包括處理廢水中之重金屬和有機物質。In one embodiment, the nanofiber membrane of the present invention can be used for wastewater treatment, filtration of bacteria or protein purification, wherein the wastewater treatment system comprises treatment of heavy metals and organic matter in the wastewater.

實例：Example: 實驗藥品Experimental drug

1.　醋酸(Riedel-Dehaen)1. Acetic acid (Riedel-Dehaen)

2.　牛血清蛋白(BSA)(Sigma)2. Bovine serum albumin (BSA) (Sigma)

3.　溶菌酶(Sigma)3. Lysozyme (Sigma)

4.　甘胺酸(Merck)4. Glycine (Merck)

5.　醋酸鈉(Merck)5. Sodium acetate (Merck)

6.　氯化鈉(Merck)6. Sodium Chloride (Merck)

7.　磷酸氫二鈉(Na2HPO4)(Merck)7. Disodium hydrogen phosphate (Na2HPO4) (Merck)

8.　磷酸二氫鈉(NaH2PO4)(Merck)8. Sodium dihydrogen phosphate (NaH2PO4) (Merck)

9.　氫氧化鈉(Merck)9. Sodium hydroxide (Merck)

10.　碳酸鈉(Riedel-Dehaen)10. Sodium carbonate (Riedel-Dehaen)

11.　GPTMS(C9 H20 O5 Si)(Merck)11. GPTMS (C 9 H 20 O 5 Si) (Merck)

12.　幾丁聚醣[(1,4)-2-胺基-2-去氧-β-D-葡聚糖](Sigma)12. Chitosan [(1,4)-2-amino-2-deoxy-β-D-glucan] (Sigma)

13.　乙二胺(Merck)13. Ethylenediamine (Merck)

14.　己二胺(ECHO)14. Hexanediamine (ECHO)

15.　酸橙(Acid Orange)7(C16 H11 N2 NaO4 S,簡稱AO7)(Sigma)15. Acid Orange 7 (C 16 H 11 N 2 NaO 4 S, referred to as AO7) (Sigma)

16.　氯化硫醯基(SOCl2 )(濃度1mol/L於二氯甲烷；東京化成工業株式會社)16. Sulfonyl chloride (SOCl 2 ) (concentration of 1 mol/L in methylene chloride; Tokyo Chemical Industry Co., Ltd.)

17.　聚丙烯腈奈米纖維膜：財團法人紡織產業綜合研究所提供17. Polyacrylonitrile nanofiber membrane: provided by the Institute of Textile Industry

18.　C ibacron Blue F3GA(Polyscience)18. C ibacron Blue F3GA (Polyscience)

19.　GTAC(Aldrich)19. GTAC (Aldrich)

20.　溴乙酸(Aldrich)20. Bromoacetic acid (Aldrich)

儀器設備equipment

1.　pH測定計(Suntex,SP-2200)1. pH meter (Suntex, SP-2200)

2.　微量電子天平(Mettler Toledo Co.,Model AG104)2. Microelectronic balance (Mettler Toledo Co., Model AG104)

3.　紫外線分光光度計(Amercham Biosciences,Ultraspec 3100pro )3. Ultraviolet spectrophotometer (Amercham Biosciences, Ultraspec 3100 pro )

4.　超純水系統(Millipore,Milli-Q)4. Ultrapure water system (Millipore, Milli-Q)

5.　高溫烘箱(Channel DV-903)5. High temperature oven (Channel DV-903)

6.　真空濃縮機(EYELA)6. Vacuum Concentrator (EYELA)

7.　水流幫浦(Aspirator,A-1000S)7. Water Flow Pump (Aspirator, A-1000S)

8.　低溫恆溫循環水槽(HCS-806)8. Low temperature constant temperature circulating water tank (HCS-806)

9.　掃流式過濾器(Stirred Cell蛋白質濃縮過濾系統)(Millipore,8200)9. Sweep filter (Stirred Cell Protein Concentration Filtration System) (Millipore, 8200)

10.　電磁加熱攪拌器(Corning,PC-420D)10. Electromagnetic heating stirrer (Corning, PC-420D)

檢測方法Detection method 1.　TBO染劑定量羧基含量1. TBO dye quantitative carboxy content

利用TBO染劑吸附量來定量待測樣本表面羧基的數量。The amount of carboxyl groups on the surface of the sample to be tested is quantified by the amount of TBO dye adsorption.

將改質過後清洗乾燥之PAN奈米纖維膜材置於6孔的容器中，並加入5 ml的1 mM TBO染劑，膜材表面羧基與染劑進行吸附反應，控制震盪速度為100 rpm。3小時後取出PAN奈米纖維膜並用去離子水清洗數次，接著再以0.1 mM NaOH溶液清洗膜材，以除去吸附在膜材表面的TBO染劑。將染色處理過之膜材置於6孔的容器中，加入5 ml醋酸水溶液，將與羧基產生離子鍵結的TBO染劑進行退染的動作。含有TBO染劑之待測溶液在紫外光-可見光波長633 nm條件下量測吸收值，並與由不同濃度之TBO溶液所得之檢量線做對照求出未知液的濃度，經換算推得膜材表面羧基的含量。The modified PAN nanofiber membrane after the modification was placed in a 6-well container, and 5 ml of 1 mM TBO dye was added, and the carboxyl group on the surface of the membrane was adsorbed with the dye to control the oscillation speed to 100 rpm. After 3 hours, the PAN nanofiber membrane was taken out and washed several times with deionized water, followed by washing the membrane with a 0.1 mM NaOH solution to remove the TBO dye adsorbed on the surface of the membrane. The dyed-treated membrane was placed in a 6-well container, and 5 ml of an aqueous acetic acid solution was added to denoise the TBO dye ion-bonded with the carboxyl group. The solution to be tested containing TBO dye is measured at an ultraviolet-visible wavelength of 633 nm, and the concentration of the unknown liquid is determined by comparison with the calibration curve obtained from different concentrations of TBO solution. The content of carboxyl groups on the surface of the material.

2.　AO7(酸橙7)染劑定量胺基含量2. AO7 (Rear 7) dye quantitative amine content

利用Acid Orange 7(AO7)染劑吸附量來定量待測樣本表面胺基的數量。The amount of the amine group on the surface of the sample to be tested is quantified by the amount of dye adsorbed by Acid Orange 7 (AO7).

將改質過後清洗乾燥完之PAN奈米纖維膜材置於6 well的容器中，並加入5 ml的1 mM AO7染劑，膜材表面以去離子水清洗數次，以除去吸附在膜材表面的染劑。將染色處理過之膜材置於6孔的容器中，加入5 ml 50 mM NaOH溶液，將與胺基產生離子鍵結的AO7染劑進行退染的動作。含有AO7染劑之待測溶液在紫外光-可見光波長485 nm條件下量測吸收值，並與由不同濃度之AO7溶液所得之檢量線做對照求出未知液的AO7濃度，經換算推得膜材表面胺基的含量。After the modification, the dried PAN nanofiber membrane was placed in a 6 well container, and 5 ml of 1 mM AO7 dye was added. The surface of the membrane was washed several times with deionized water to remove the adsorption on the membrane. The dye on the surface. The dyed treated membrane was placed in a 6-well container, and 5 ml of a 50 mM NaOH solution was added to deproteinize the AO7 dye which is ion-bonded with the amine group. The solution to be tested containing AO7 dye is measured at the ultraviolet-visible wavelength of 485 nm, and the AO7 concentration of the unknown solution is determined by comparison with the calibration curve obtained from different concentrations of AO7 solution. The content of amine groups on the surface of the membrane.

3.　奈米纖維膜固定化Cibacron Blue F3GA含量之測定3. Determination of the content of Cibacron Blue F3GA immobilized on nanofiber membrane

將具有反應性染料Cibacron Blue F3GA之待測樣品置於紫外光分光光度計中，並以OD 612 nm條件下量測吸收值，進而獲得奈米纖維膜固定化染料的含量。The sample to be tested having the reactive dye Cibacron Blue F3GA was placed in an ultraviolet spectrophotometer, and the absorption value was measured at OD 612 nm, thereby obtaining the content of the nanofiber membrane-immobilized dye.

4.　蛋白質濃度測定(紫外光吸光法)4. Determination of protein concentration (UV light absorption method)

胺基酸的芳香基團在280 nm有吸光，蛋白質胜鏈骨架上的基團在280 nm附近有吸光。由於各種蛋白質所含芳香族胺基酸組成不一，因此在280 nm的吸光能力亦不同，可以分子消光係數(molar extinction coefficient)來表示。The aromatic group of the amino acid absorbs light at 280 nm, and the group on the protein chain backbone absorbs light around 280 nm. Since various proteins contain different aromatic amino acids, the absorbance at 280 nm is also different and can be expressed by the molecular extinction coefficient.

1.　將牛血清蛋白標準溶液(2 mg/ml)做系列稀釋，然後再放入紫外光-可見光分光光譜儀中，以波長280 nm測定之，代入下列公式即可求得牛血清蛋白真實濃度(mg/ml)(Peters,1985)。1. Serially dilute the bovine serum albumin standard solution (2 mg/ml), then place it in an ultraviolet-visible spectrophotometer at a wavelength of 280 nm and substitute the following formula to determine the true concentration of bovine serum albumin ( Mg/ml) (Peters, 1985).

2.　將溶菌酶標準溶液(2 mg/ml)做系列稀釋，然後再放入紫外光-可見光分光光譜儀中，以波長280 nm測定之，代入下列公式即可求得溶菌酶真實濃度(mg/ml)。2. The lysozyme standard solution (2 mg/ml) is serially diluted, and then placed in an ultraviolet-visible spectrophotometer at a wavelength of 280 nm. Substituting the following formula can determine the true concentration of lysozyme (mg/ Ml).

5.　蛋白質吸附試驗5. Protein adsorption test

以20 mM緩衝溶液(醋酸鹽緩衝溶液，pH 4~5；磷酸鹽緩衝溶液，pH 6~8；甘胺酸-NaOH，pH 9~10；碳酸鹽緩衝溶液，pH 11~12)配製牛血清蛋白溶液(2 mg/ml)或溶菌酶溶液(2 mg/ml)。將待測奈米纖維膜置於上述所配製之牛血清蛋白溶液或溶菌酶溶液中控制震盪速度為100 rpm，在常溫下連續作用3小時；移除反應過後之奈米纖維膜；接著在紫外光-可見光分光光譜儀中波長280 nm下測定殘留液中牛血清蛋白或溶菌酶濃度(mg/ml)，由吸附前後蛋白質濃度之變化計算薄膜吸附量。Prepare bovine serum with 20 mM buffer solution (acetate buffer solution, pH 4~5; phosphate buffer solution, pH 6~8; glycine-NaOH, pH 9~10; carbonate buffer solution, pH 11~12) Protein solution (2 mg/ml) or lysozyme solution (2 mg/ml). The nanofiber membrane to be tested is placed in the bovine serum albumin solution or the lysozyme solution prepared above to control the oscillation speed to 100 rpm, and continuously operated at normal temperature for 3 hours; the nanofiber membrane after the reaction is removed; The concentration of bovine serum albumin or lysozyme (mg/ml) in the residual liquid was measured at 280 nm in a light-visible spectrophotometer, and the adsorption amount of the film was calculated from the change of protein concentration before and after adsorption.

實例1經乙二胺改質之PAN奈米纖維膜對AO7吸附效能的影響試驗Example 1 Effect of PAN Nanofiber Membrane Modified by Ethylenediamine on Adsorption Efficiency of AO7

先將PAN奈米纖維膜裁切成約2cmx2cm大小尺寸，以微量電子天平精稱其單片膜材之重量並記錄(每片膜材約0.02 g)。接者，使PAN奈米纖維膜分別接受100%(體積/體積)乙二胺溶液於30、50、70、80及90℃條件下處理0.5、1.0、2.0與3.0小時。緊接者，將改質後的PAN奈米纖維膜片置入5 ml之AO7於0.1mM HCl溶液之水溶液中吸附3 hr後觀測其吸附值的變化，並計算PAN奈米纖維膜吸附AO7染劑量。。再以所測得之AO7濃度(umol/g)分別對反應溫度及時間作圖(如圖1所示)。The PAN nanofiber membrane was first cut into a size of about 2 cm x 2 cm, and the weight of the monolithic membrane was weighed and recorded with a microelectronic balance (about 0.02 g per membrane). Next, the PAN nanofiber membrane was subjected to treatment with 100% (v/v) ethylenediamine solution at 30, 50, 70, 80 and 90 ° C for 0.5, 1.0, 2.0 and 3.0 hours, respectively. Immediately after the modified PAN nanofiber membrane was placed in 5 ml of AO7 in an aqueous solution of 0.1 mM HCl solution for 3 hr, the change of adsorption value was observed, and the PAN nanofiber membrane adsorption AO7 dyeing was calculated. dose. . The measured AO7 concentration (umol/g) was then plotted against the reaction temperature and time (as shown in Figure 1).

從圖1顯示，當反應溫度增加時，PAN奈米纖維膜對AO7的吸附量有明顯的上升趨勢，在90℃反應溫度下達最大值；以及隨著反應時間的增加，PAN奈米纖維膜對AO7的吸附量亦有明顯上升的趨勢，且在3小時處理溫度下達最大值。It is shown in Fig. 1 that when the reaction temperature is increased, the adsorption amount of PAN nanofiber membrane to AO7 has a significant upward trend, reaching a maximum at a reaction temperature of 90 ° C; and as the reaction time increases, the PAN nanofiber membrane pair The adsorption capacity of AO7 also showed a significant upward trend and reached a maximum at the 3-hour treatment temperature.

綜合上述，本試驗在反應時間3小時與反應溫度90℃條件下可達AO7最大吸附量，顯示在此條件下PAN奈米纖維膜材表面胺基離子鍵結的能力可達最高。Based on the above, the maximum adsorption capacity of AO7 can be reached in the reaction time of 3 hours and the reaction temperature of 90 °C, which shows that the ability of the surface amino group ion bonding of PAN nanofiber membrane can reach the highest under this condition.

實real 例2PAN奈米纖維膜以乙二胺或己二胺改質後AO7吸附效能的比較Example 2 Comparison of adsorption efficiency of AO7 after modification of PAN nanofiber membrane with ethylenediamine or hexamethylenediamine

首先將PAN奈米纖維膜裁切成約2cmx2cm大小尺寸，以微量電子天平精稱其單片膜材之重量並記錄(每片膜材約0.02 g)；將PAN奈米纖維膜置於3M NaOH溶液中於85℃下反應30分鐘；隨後將經處理之PAN奈米纖維膜在置於0.1M HCl溶液中於室溫下過夜，以獲得經羧基改質之PAN奈米纖維膜；接著再將該PAN奈米纖維膜置於1M SOCl2 溶液中於60℃下反應30分鐘以獲得經醯氯基改質之PAN奈米纖維膜。First, the PAN nanofiber membrane was cut into a size of about 2 cm x 2 cm, and the weight of the monolithic membrane was weighed and recorded by a microelectronic balance (about 0.02 g per membrane); the PAN nanofiber membrane was placed in 3 M NaOH. The solution was reacted at 85 ° C for 30 minutes; then the treated PAN nanofiber membrane was placed in a 0.1 M HCl solution at room temperature overnight to obtain a carboxyl-modified PAN nanofiber membrane; The PAN nanofiber membrane was subjected to a reaction in a 1 M SOCl 2 solution at 60 ° C for 30 minutes to obtain a ruthenium chloride-modified PAN nanofiber membrane.

將100%(體積/體積)乙二胺溶液以及100%(體積/體積)己二胺溶液分別以去離子水配製成50%(體積/體積)乙二胺溶液及50%(體積/體積)己二胺溶液。100% (v/v) ethylenediamine solution and 100% (v/v) hexamethylenediamine solution were respectively prepared into 50% (v/v) ethylenediamine solution and 50% (vol/vol) in deionized water. a hexamethylene diamine solution.

將所獲得含醯氯基之PAN奈米纖維膜與未改質之PAN奈米纖維膜分別與乙二胺溶液(100%(體積/體積)與50%(體積/體積))及己二胺溶液(100%(體積/體積)與50%(體積/體積))在90℃環境下，反應3小時。反應完成後，將上述PAN奈米纖維膜片取出以去離子水清洗並去除多餘的水分，接著將膜片置入5 ml AO7水溶液中，震盪速度維持在100 rpm，在室溫下進行吸附反應，3 hr後觀測其變化並計算經改質的PAN奈米纖維膜之AO7吸附量，計算結果如表1所示。The obtained PAN nanofiber-containing PAN nanofiber membrane and the unmodified PAN nanofiber membrane and the ethylenediamine solution (100% (vol/vol) and 50% (vol/vol)) and hexamethylenediamine, respectively The solution (100% (vol/vol) and 50% (vol/vol)) was reacted for 3 hours at 90 °C. After the reaction was completed, the above PAN nanofiber membrane was taken out and washed with deionized water to remove excess water, and then the membrane was placed in a 5 ml aqueous solution of AO7, the oscillation speed was maintained at 100 rpm, and the adsorption reaction was carried out at room temperature. After 3 hr, the change was observed and the amount of AO7 adsorption of the modified PAN nanofiber membrane was calculated. The calculation results are shown in Table 1.

由表1的結果可看出，經NaOH與SOCl2 處理過之PAN奈米纖維膜片，在經過濃度100%(體積/體積)與50%(體積/體積)的乙二胺或己二胺溶液反應後，其AO7吸附量大多在120 μeq/g以上，其中以50%乙二胺處理之反應，其吸附量僅達99 μeq/g左右。反觀直接與乙二胺或己二胺改質的PAN奈米纖維膜對AO7的吸附效果，除100%乙二胺外，其餘操作模式之吸附量呈現較低之狀況，僅有30～50 μeq/g。整體而言，直接以100%(體積/體積)乙二胺溶液處理之PAN奈米纖維膜材表面胺基含量與經NaOH與SOCl2 處理過之PAN奈米纖維膜片無太大差異(表1)。It can be seen from the results in Table 1 that the PAN nanofiber membrane treated with NaOH and SOCl 2 is subjected to a concentration of 100% (v/v) and 50% (v/v) of ethylenediamine or hexamethylenediamine. After the solution reaction, the adsorption amount of AO7 is mostly above 120 μeq/g, and the adsorption amount of the reaction with 50% ethylenediamine is only about 99 μeq/g. In contrast, the adsorption effect of PAN nanofiber membrane directly modified with ethylenediamine or hexamethylenediamine on AO7, except for 100% ethylenediamine, the adsorption amount of other modes of operation is lower, only 30-50 μeq /g. Overall, the surface amine content of PAN nanofiber membrane directly treated with 100% (v/v) ethylenediamine solution is not much different from that of PAN nanofiber membrane treated with SOC and SOCl 2 (Table) 1).

實例3離子交換型之奈米纖維膜吸附蛋白質(BSA)試驗Example 3 ion exchange type nanofiber membrane adsorption protein (BSA) test

以20 mM緩衝溶液(醋酸鹽緩衝溶液，pH 4~5；磷酸鹽緩衝溶液，pH 6~8；甘胺酸-NaOH，pH 9~10；碳酸鹽緩衝溶液，pH 11~12)配製pH 4-12之牛血清蛋白溶液(2 mg/ml)。Prepare pH 4 with 20 mM buffer solution (acetate buffer solution, pH 4~5; phosphate buffer solution, pH 6~8; glycine-NaOH, pH 9~10; carbonate buffer solution, pH 11~12) -12 bovine serum albumin solution (2 mg/ml).

將實例2所獲得直接以100%(體積/體積)乙二胺改質之PAN奈米纖維膜與Sartobind市售商業膜(Sartobind C膜含弱酸R-COO-；Sartobind D膜含R-CH2 N+ (C2 H5 )2 ；Sartobind Q膜含R-CH2 N+ (CH3 )3 ；以及Sartobind S膜含R-CH2 SO3 - )置於室溫下pH 4-12之BSA溶液中進行吸附反應3 hr，其中震盪速度維持在100 rpm。所得結果如圖2所示。The PAN nanofiber membrane obtained by directly modifying 100% (vol/vol) ethylenediamine obtained in Example 2 and the commercially available commercial membrane of Sartobind (Sartobind C membrane containing weak acid R-COO-; Sartobind D membrane containing R-CH 2 N + (C 2 H 5 ) 2 ; Sartobind Q film containing R-CH 2 N + (CH 3 ) 3 ; and Sartobind S film containing R-CH 2 SO 3 - ) BSA at room temperature, pH 4-12 The adsorption reaction was carried out for 3 hr in the solution, wherein the shaking speed was maintained at 100 rpm. The results obtained are shown in Figure 2.

從圖2的趨勢可看出，改質後的PAN奈米纖維膜對BSA的吸附量隨著pH上升而增加，實驗發現在中性偏弱鹼環境下吸附效果較好，在pH 9時其BSA的吸附量高達80 mg/g。換言之，1克重的PAN奈米纖維膜片在pH 9環境下，可以吸附80 mg左右的BSA。當pH在11-12時，BSA吸附的趨勢轉為下降。因此，就每單位膜重量而言，本發明直接以100%(體積/體積)乙二胺改質之PAN奈米纖維薄膜的吸附效率較商業化膜更佳。It can be seen from the trend of Fig. 2 that the adsorption amount of BSA on the modified PAN nanofiber membrane increases with the increase of pH. It is found that the adsorption effect is better under neutral weak alkaline environment, and its pH is 9 at pH 9. The adsorption capacity of BSA is as high as 80 mg/g. In other words, a 1 gram PAN nanofiber membrane can adsorb about 80 mg of BSA at pH 9. When the pH is between 11 and 12, the tendency of BSA adsorption turns to decrease. Therefore, the adsorption efficiency of the PAN nanofiber film directly modified with 100% (v/v) ethylenediamine of the present invention is better than that of the commercial film per unit weight of the film.

實例4　經不同濃度幾丁聚醣與不同濃度GPTMS改質之(-COOH)奈米纖維膜Example 4 (-COOH) nanofiber membrane modified with different concentrations of chitosan and different concentrations of GPTMS

將實例2所獲得之經羧基改質之PAN奈米纖維膜分別置於80℃下幾丁聚醣溶液(濃度為0.1%~1%(重量/體積))與幾丁聚醣/GPTMS錯合物溶液(其中幾丁聚醣濃度為0.1%~1%(重量/體積)，GPTMS濃度為1%~70%(體積/體積))中反應3小時，接著進行所獲得經改質之PAN奈米薄膜對AO7吸附效能的比較。The carboxylated PAN nanofiber membrane obtained in Example 2 was placed at 80 ° C in a chitosan solution (concentration of 0.1% to 1% (w/v)) and chitosan/GPTMS was mismatched. The solution (in which the chitosan concentration is 0.1% to 1% (w/v), the GPTMS concentration is 1% to 70% (vol/vol)), the reaction is carried out for 3 hours, and then the modified PAN naf obtained is obtained. Comparison of the adsorption performance of rice film on AO7.

圖3之實驗結果顯示，從莫耳數來看在70% GPTMS下，不同濃度的幾丁聚醣對AO7的吸附量為20 μmol/g；從吸附的毫克數來看在70% GPTMS下不同濃度的幾丁聚醣對AO7的吸附量為7 mg/g，並不有太大的差異。因此，GPTMS濃度越高有助於幾丁聚醣固定在奈米纖維膜上越多。The experimental results in Figure 3 show that, under the condition of mole number, the adsorption of AO7 by different concentrations of chitosan is 20 μmol/g under 70% GPTMS; the difference in the number of milligrams of adsorption is different under 70% GPTMS. The adsorption amount of chitosan to AO7 was 7 mg/g, which was not much different. Therefore, the higher the concentration of GPTMS, the more the chitosan is immobilized on the nanofiber membrane.

實例5經不同濃度幾丁聚醣與不同濃度GPTMS改質之(-COCl)奈米纖維膜Example 5 (-COCl) nanofiber membrane modified with different concentrations of chitosan and different concentrations of GPTMS

將實例2所獲得之經醯氯基改質之PAN奈米纖維膜分別置於80℃下幾丁聚醣溶液(濃度為0.1%~1%(重量/體積))與幾丁聚醣/GPTMS錯合物溶液(其中幾丁聚醣濃度為0.1%~1%(重量/體積)，GPTMS濃度為1%~70%(體積/體積))中反應3小時，接著進行所獲得經改質之PAN奈米薄膜對AO7與TBO吸附效能的比較。The ruthenium chloride-modified PAN nanofiber membrane obtained in Example 2 was placed in a chitosan solution (concentration of 0.1% to 1% (w/v)) and chitosan/GPTMS at 80 ° C, respectively. The complex solution (in which the chitosan concentration is 0.1% to 1% (w/v), the GPTMS concentration is 1% to 70% (vol/vol)), the reaction is carried out for 3 hours, and then the obtained modified product is obtained. Comparison of the adsorption performance of PAN nanofilms on AO7 and TBO.

圖4顯示實驗中AO7是測定膜上的胺基數量，結果顯示從莫耳數來看在1%幾丁聚醣添加少量的GPTMS可增加對AO7的吸附量，為80 μmol/g；從吸附的毫克數來看在1%幾丁聚醣添加少量的GPTMS對AO7的吸附量為35 mg/g。此外，實驗證實少量GPTMS可幫助幾丁聚醣固定在膜上越多，但與改質膜未添加GPTMS的幾丁聚醣吸附量相差1.5倍。Figure 4 shows that AO7 is the number of amine groups on the membrane. The results show that the addition of a small amount of GPTMS to 1% chitosan can increase the adsorption amount of AO7 to 80 μmol/g from the molar number. The number of milligrams was observed to increase the adsorption amount of AO7 to 35 mg/g by adding a small amount of GPTMS to 1% chitosan. In addition, experiments have confirmed that a small amount of GPTMS can help the chitosan to be immobilized on the membrane, but the amount of chitosan adsorbed by the modified membrane without adding GPTMS is 1.5 times.

實驗中TBO是測定膜上羧基的數量，結果顯示數據皆為負值，表示膜上的羧基都已完全反應。In the experiment, TBO was used to determine the number of carboxyl groups on the membrane. The results showed that the data were all negative, indicating that the carboxyl groups on the membrane were completely reacted.

實例6以反應性染料(Cibacron Blue F3GA)固定化金屬親和性薄膜對蛋白質吸附效能之影響Example 6 Effect of Immobilized Metal Affinity Film Immobilized with Reactive Dyes (Cibacron Blue F3GA) on Protein Adsorption Efficiency

先配製10 ml Cibacron blue F3GA(0.25 mg/ml)溶解於水之溶液，再加入2 ml 20% NaCl(重量/體積)混合均勻備用。First prepare 10 ml of Cibacron blue F3GA (0.25 mg/ml) dissolved in water, then add 2 ml of 20% NaCl (weight/volume) and mix well.

6.1　經幾丁聚醣、乙二胺或己二胺改質之奈米纖維膜固定Cibacron Blue F3GA之能力比較6.1 Comparison of the ability of nanofiber membrane modified by chitosan, ethylenediamine or hexamethylene diamine to fix Cibacron Blue F3GA

將實例2所獲得之經醯氯基改質之PAN奈米纖維膜分別置於80℃下幾丁聚醣溶液(濃度為0.1%、0.2%、0.5%及1%(重量/體積))中反應3小時。將所獲得4種經改質之奈米纖維膜以及由實例2所獲得經NaOH、SOCl2 及100%EDA或100%HDA改質之奈米纖維膜加入5 ml Cibacron blue F3GA/NaCl溶液，並於60℃下反應1小時；隨後再加入100 mg Na2 CO3 待完全溶解後，在60℃反應3 hr；以及將殘餘染料以去離子水多次洗淨，其中Cibacron Blue F3GA係經由其三氮六環中的氯與乙二胺或己二胺的胺基產生親核性反應。The ruthenium chloride-modified PAN nanofiber membrane obtained in Example 2 was placed in a chitosan solution (concentration of 0.1%, 0.2%, 0.5%, and 1% (weight/volume)) at 80 ° C, respectively. Reaction for 3 hours. The obtained four modified nanofiber membranes and the nanofiber membrane modified by NaOH, SOCl 2 and 100% EDA or 100% HDA obtained in Example 2 were added to 5 ml of Cibacron blue F3GA/NaCl solution, and The reaction was carried out at 60 ° C for 1 hour; then, after further adding 100 mg of Na 2 CO 3 to be completely dissolved, the reaction was carried out at 60 ° C for 3 hr; and the residual dye was washed several times with deionized water, wherein Cibacron Blue F3GA was passed through three The chlorine in the nitrogen hexacyclic ring reacts with the amine group of ethylenediamine or hexamethylenediamine.

從圖5的數據可看出幾丁聚醣對Cibacron Blue F3GA的吸附效果較低，且實際上可用肉眼辨別奈米纖維膜上有淡淡的藍色；而EDA或HDA對Cibacron Blue F3GA的吸附效果較佳，且實際上奈米纖維膜反應完會變成深藍色的膜片。From the data in Figure 5, it can be seen that chitosan has a low adsorption effect on Cibacron Blue F3GA, and actually can visually distinguish the light blue color on the nanofiber membrane; and the adsorption effect of EDA or HDA on Cibacron Blue F3GA. Preferably, and in fact, the nanofiber membrane is turned into a dark blue membrane upon reaction.

6.2經幾丁聚醣、乙二胺或己二胺改質之奈米纖維膜固定Cibacron Blue F3GA之能力比較6.2 Comparison of the ability of nanofiber membrane modified by chitosan, ethylenediamine or hexamethylene diamine to fix Cibacron Blue F3GA

將實例7.1之經1%(重量/體積)幾丁聚醣、經1%(重量/體積)幾丁聚醣與Cibacron Blue F3GA以及經EDA與Cibacron Blue F3GA改質之奈米纖維膜分別置於0.1M CuSO4 、CoSO4 、NiSO4 與ZnSO4 溶液中反應3小時，接著將所獲得之固定化金屬親和性薄膜進行吸附BSA或溶菌酶試驗(BSA的pH值為5以及溶菌酶的pH值為7)，其係以二重覆方式，並觀察其吸附量。實驗結果如圖6及34所示。The 1% (w/v) chitosan of Example 7.1, the 1% (w/v) chitosan and Cibacron Blue F3GA, and the EDA and Cibacron Blue F3GA modified nanofiber membrane were placed separately 0.1M CuSO 4 , CoSO 4 , NiSO 4 and ZnSO 4 solution were reacted for 3 hours, and then the obtained immobilized metal affinity film was subjected to adsorption BSA or lysozyme test (pH of BSA was 5 and pH of lysozyme) For 7), it is in a double-over manner and the amount of adsorption is observed. The experimental results are shown in Figures 6 and 34.

圖6顯示單純幾丁聚醣固定化四種金屬吸附BSA，固定銅薄膜並未吸附BSA，固定鎳與鋅的薄膜，吸附BSA效果差不多，為35.34 mg/g(0.16 mg/cm2 )；而幾丁聚醣與染料做結合並分別固定化四種金屬均無法吸附BSA；EDA與染料做結合固定化金屬也是以鎳吸附量較高，為52.21 mg/g(0.23 mg/cm2 )。Figure 6 shows that the immobilized copper film immobilized on the four metals adsorbed BSA, the fixed copper film did not adsorb BSA, and the nickel and zinc films were fixed, and the effect of adsorbing BSA was about 35.34 mg/g (0.16 mg/cm 2 ). The combination of chitosan and dyes and the immobilization of four metals can not adsorb BSA; the combined immobilization of EDA and dye is also higher than that of nickel, which is 52.21 mg/g (0.23 mg/cm 2 ).

圖7顯示單純以幾丁聚醣固定化金屬以鎳的效果最好，為110.22 mg/g(0.50 mg/cm2 )；幾丁聚醣與染料做結合固定化金屬中鎳對溶菌酶的吸附最好，為140.63 mg/g(0.586 mg/cm2 )；而EDA與染料結合固定化金屬以鋅的效果最好，是這次反應性染料實驗中吸附值最高者，為143.58 mg/g(0.587 mg/cm2 )。Figure 7 shows that the effect of immobilizing metal with chitosan is best with nickel, which is 110.22 mg/g (0.50 mg/cm 2 ). The adsorption of lysozyme by nickel in the immobilized metal combined with chitosan and dye Preferably, it is 140.63 mg/g (0.586 mg/cm 2 ); while EDA and dye combined with immobilized metal have the best effect on zinc, which is the highest adsorption value in this reactive dye experiment, which is 143.58 mg/g (0.587 Mg/cm 2 ).

因此，在批次法下，Cibacron Blue F3GA固定在奈米纖維膜上的量會影響到對蛋白質的吸附效果。Therefore, under the batch method, the amount of Cibacron Blue F3GA immobilized on the nanofiber membrane affects the adsorption of protein.

實例7以GTAC改質對AO7與TBO染劑吸附效能之影響Example 7 Effect of GTAC Modification on Adsorption Efficiency of AO7 and TBO Dyes

將實例2所獲得之經醯氯基改質之PAN奈米纖維膜置於80℃下幾丁聚醣溶液(濃度為0.5%(重量/體積))中反應3小時。分別加入2.5ml GTAC溶液至所獲得經幾丁聚醣改質之奈米纖維膜以及由實例2所獲得經NaOH、SOCl2 及100%EDA或100%HDA改質之奈米纖維膜中，並放置於60℃烘箱中反應3 hr；接著以去離子水多次沖洗乾淨後，進行AO7與TBO吸附實驗。實驗結果如圖8所示。The ruthenium chloride-modified PAN nanofiber membrane obtained in Example 2 was placed in a chitosan solution (concentration: 0.5% (w/v)) at 80 ° C for 3 hours. Adding 2.5 ml of GTAC solution to the obtained chitosan-modified nanofiber membrane and the nanofiber membrane modified by NaOH, SOCl 2 and 100% EDA or 100% HDA obtained in Example 2, and The reaction was carried out in an oven at 60 ° C for 3 hr; then, after washing with deionized water several times, AO7 and TBO adsorption experiments were carried out. The experimental results are shown in Figure 8.

AO7吸附實驗結果顯示，(1)改質完的幾丁聚醣對AO7染料的吸附量為119.29 μmol/g；(2)改質完的EDA對AO7染料的吸附量為262.65 μmol/g；(1)改質完的HDA對AO7染料的吸附量為273.48 μmol/g。證實這個改質方式確實可大大增加膜上的胺基，吸附量高達250 μmol/g以上(圖8)。The results of AO7 adsorption experiments showed that (1) the amount of modified galvanose adsorbed to AO7 dye was 119.29 μmol/g; (2) the amount of modified EDA adsorbed to AO7 dye was 262.65 μmol/g; 1) The amount of adsorbed HDA to AO7 dye was 273.48 μmol/g. It was confirmed that this modification method can greatly increase the amine group on the membrane, and the adsorption amount is as high as 250 μmol/g or more (Fig. 8).

TBO吸附實驗結果顯示數據皆為負值，表示膜上的羧基都已完全反應。The results of the TBO adsorption experiment showed that the data were all negative, indicating that the carboxyl groups on the membrane were completely reacted.

實例8以GTAC改質之薄膜對蛋白質吸附效能之影響Example 8 Effect of GTAC Modified Film on Protein Adsorption Efficiency

將實例2所獲得之經醯氯基改質之PAN奈米纖維膜置於80℃下幾丁聚醣溶液(濃度為0.5%(重量/體積))中反應3小時。分別加入2.5ml GTAC溶液至所獲得經幾丁聚醣改質之奈米纖維膜以及由實例2所獲得經NaOH、SOCl2 及100%EDA或100%HDA改質之奈米纖維膜中，並放置於60℃烘箱中反應3 hr；接著以去離子水多次沖洗乾淨後，進行BSA吸附實驗(因經改質之薄膜上尾端帶有正電荷)。The ruthenium chloride-modified PAN nanofiber membrane obtained in Example 2 was placed in a chitosan solution (concentration: 0.5% (w/v)) at 80 ° C for 3 hours. Adding 2.5 ml of GTAC solution to the obtained chitosan-modified nanofiber membrane and the nanofiber membrane modified by NaOH, SOCl 2 and 100% EDA or 100% HDA obtained in Example 2, and The reaction was carried out in an oven at 60 ° C for 3 hr; then, after washing with deionized water several times, a BSA adsorption experiment was performed (because the modified film had a positive charge on the tail end).

本實驗吸附BSA的pH值為7，以二重覆方式進行實驗。圖9顯示(1)改質後的幾丁聚醣對BSA的吸附量為49.22 mg/g(0.22 mg/cm2 )；(2)改質後的EDA對BSA的吸附量為83.13 mg/g(0.35 mg/cm2 )；(3)改質後的HDA對BSA的吸附量為51.80 mg/g(0.29 mg/cm2 )。In this experiment, the pH of the adsorbed BSA was 7, and the experiment was carried out in a double-over manner. Figure 9 shows that (1) the amount of modified chitosan adsorbed to BSA was 49.22 mg/g (0.22 mg/cm 2 ); (2) the amount of modified EDA to BSA was 83.13 mg/g. (0.35 mg/cm 2 ); (3) The amount of BSA adsorbed by the modified HDA was 51.80 mg/g (0.29 mg/cm 2 ).

實例9以溴乙酸改質的PAN膜對蛋白質吸附效能之影響Example 9 Effect of PAN Membranes Modified with Bromoacetic Acid on Protein Adsorption Efficiency

將50ml 2M NaOH、50ml 1M碳酸氫鈉與250mg溴乙酸混合，以獲得一具有總體積為100ml之溴乙酸溶液。50 ml of 2 M NaOH, 50 ml of 1 M sodium hydrogencarbonate and 250 mg of bromoacetic acid were mixed to obtain a bromoacetic acid solution having a total volume of 100 ml.

將實例2所獲得之經醯氯基改質之PAN奈米纖維膜置於80℃下幾丁聚醣溶液(濃度為0.5%(重量/體積))中反應3小時。分別加入2.5ml溴乙酸溶液至所獲得經幾丁聚醣改質之奈米纖維膜以及由實例2所獲得經NaOH、SOCl2 及100%EDA或100%HDA改質之奈米纖維膜中，並放置於60℃烘箱中反應3 hr；接著以去離子水多次沖洗乾淨後，進行溶菌酶吸附實驗(因經改質之薄膜上尾端帶有負電荷(-COO- ))。The ruthenium chloride-modified PAN nanofiber membrane obtained in Example 2 was placed in a chitosan solution (concentration: 0.5% (w/v)) at 80 ° C for 3 hours. Adding 2.5 ml of bromoacetic acid solution to the obtained chitosan-modified nanofiber membrane and the nanofiber membrane modified by NaOH, SOCl 2 and 100% EDA or 100% HDA obtained in Example 2, And placed in an oven at 60 ° C for 3 hr; then rinsed with deionized water several times, and then lysozyme adsorption experiment (because the modified film has a negative charge (-COO - ) at the end).

本實驗吸附溶菌酶的pH值為7，以二重覆方式進行實驗。圖10結果顯示(1)改質後幾丁聚醣對溶菌酶的吸附量為168.19 mg/g(0.79 mg/cm2)；(2)改質後的EDA對溶菌酶的吸附量為43.24 mg/g(0.24 mg/cm2)；(3)改質後的HDA對溶菌酶無吸附作用產生。In this experiment, the pH of the adsorbed lysozyme was 7, and the experiment was carried out in a double-over manner. The results in Figure 10 show that (1) the adsorption amount of chitosan to lysozyme after modification is 168.19 mg/g (0.79 mg/cm2); (2) the adsorption capacity of modified EDA to lysozyme is 43.24 mg/ g (0.24 mg/cm2); (3) The modified HDA produced no adsorption to lysozyme.

實例10以溴乙酸改質固定化金屬螯合對吸附蛋白質影響Example 10 Effect of Modification of Immobilized Metal Chelate on Adsorption of Protein by Bromoacetic Acid

將50ml 2M NaOH、50ml 1M碳酸氫鈉與250mg溴乙酸混合，以獲得一具有總體積為100ml之溴乙酸溶液。50 ml of 2 M NaOH, 50 ml of 1 M sodium hydrogencarbonate and 250 mg of bromoacetic acid were mixed to obtain a bromoacetic acid solution having a total volume of 100 ml.

將實例2所獲得之經醯氯基改質之PAN奈米纖維膜置於80℃下幾丁聚醣溶液(濃度為0.5%(重量/體積))中反應3小時。分別加入2.5ml溴乙酸溶液至所獲得經幾丁聚醣改質之奈米纖維膜以及由實例2所獲得經NaOH、SOCl2 及100%EDA或100%HDA改質之奈米纖維膜中，並放置於60℃烘箱中反應3 hr；接著以去離子水多次沖洗乾淨。將上述改質後奈米纖維膜分別置於0.1M CuSO4 、CoSO4 、NiSO4 和ZnSO4 溶液中反應3hr以進行固定化，接著將所得奈米纖維膜樣品進行BSA及溶菌酶吸附試驗，觀察其吸附量。The ruthenium chloride-modified PAN nanofiber membrane obtained in Example 2 was placed in a chitosan solution (concentration: 0.5% (w/v)) at 80 ° C for 3 hours. Adding 2.5 ml of bromoacetic acid solution to the obtained chitosan-modified nanofiber membrane and the nanofiber membrane modified by NaOH, SOCl 2 and 100% EDA or 100% HDA obtained in Example 2, And placed in an oven at 60 ° C for 3 hr; then rinsed several times with deionized water. The modified nanofiber membrane was separately placed in a 0.1 M CuSO 4 , CoSO 4 , NiSO 4 and ZnSO 4 solution for 3 hr to be immobilized, and then the obtained nanofiber membrane sample was subjected to BSA and lysozyme adsorption test. Observe the amount of adsorption.

本實驗吸附BSA的pH質為5，以二重覆方式進行實驗。圖11顯示固定化銅在這三種改質方式中都無法吸附BSA，固定化鎳在這三種改質方式中會有比較好的吸附量。幾丁聚醣固定化鎳吸附量為56.48 mg/g(0.27 mg/cm2 )；EDA固定化鎳吸附量為23.92 mg/g(0.11 mg/cm2 )；HDA固定化鎳吸附量為30.88 mg/g(0.13 mg/cm2 )。In this experiment, the pH of the adsorbed BSA was 5, and the experiment was carried out in a double-over manner. Figure 11 shows that the immobilized copper is unable to adsorb BSA in all three modification modes, and the immobilized nickel has a relatively good adsorption amount in these three modification modes. The adsorption capacity of chitosan-immobilized nickel was 56.48 mg/g (0.27 mg/cm 2 ); the adsorption capacity of EDA-immobilized nickel was 23.92 mg/g (0.11 mg/cm 2 ); the adsorption of HDA-immobilized nickel was 30.88 mg. /g (0.13 mg/cm 2 ).

本實驗吸附溶菌酶的pH質為7，以二重覆方式進行實驗。圖12顯示幾丁聚醣固定化鈷吸附量為121.48 mg/g(0.60 mg/cm2)；EDA固定化鈷吸附量為270.15 mg/g(1.10 mg/cm2)；HDA固定化銅吸附量為51.39 mg/g(0.25 mg/cm2)。In this experiment, the pH of the adsorbed lysozyme was 7, and the experiment was carried out in a double-over manner. Figure 12 shows that the adsorption of chitosan-immobilized cobalt is 121.48 mg/g (0.60 mg/cm2); the adsorption capacity of EDA-immobilized cobalt is 270.15 mg/g (1.10 mg/cm2); the adsorption capacity of HDA-immobilized copper is 51.39. Mg/g (0.25 mg/cm2).

實例11改質奈米纖維膜對BSA及溶菌酶的吸附效率之比較試驗(批次法)Example 11 Comparison of adsorption efficiency of BSA and lysozyme by modified nanofiber membrane (batch method)

以20 mM pH7之磷酸鹽緩衝溶液配製牛血清蛋白溶液(2 mg/ml)或溶菌酶溶液(2 mg/ml)。將表2所示之待測奈米纖維膜置於上述所配製之牛血清蛋白溶液或溶菌酶溶液中控制震盪速度為100 rpm，在常溫下連續作用3小時；移除反應過後之奈米纖維膜；接著在紫外光-可見光分光光譜儀中波長280 nm下測定殘留液中牛血清蛋白或溶菌酶濃度(mg/ml)，由吸附前後蛋白質濃度之變化計算薄膜吸附量。表2顯示本發明之奈米纖維膜對於BSA及溶菌酶之吸附能力。A bovine serum albumin solution (2 mg/ml) or a lysozyme solution (2 mg/ml) was prepared in a 20 mM pH 7 phosphate buffer solution. The nanofiber membrane to be tested shown in Table 2 was placed in the above prepared bovine serum albumin solution or lysozyme solution to control the oscillation speed to 100 rpm, and continuously operated at normal temperature for 3 hours; the nanofiber after the reaction was removed. The membrane was then measured for the concentration of bovine serum albumin or lysozyme (mg/ml) in the residual liquid at a wavelength of 280 nm in an ultraviolet-visible spectrophotometer. The amount of membrane adsorption was calculated from the change in protein concentration before and after adsorption. Table 2 shows the adsorption ability of the nanofiber membrane of the present invention for BSA and lysozyme.

實例12以PAN改質膜從新鮮雞蛋白中純化溶菌酶試驗(掃流式法)Example 12 Purification of Lysozyme from Fresh Chicken Protein by PAN Modified Membrane (Sweep Method)

本實驗目的是將表面改質的奈米纖維膜，以市售的掃流式過濾器進行溶菌酶的分離純化。選擇批次法對溶菌酶吸附效率較佳的改質薄膜進行掃流式蛋白質純化試驗，並將改質薄膜純化效率做比較。The purpose of this experiment was to separate and purify the lysozyme from a surface-modified nanofiber membrane using a commercially available sweep filter. The batch method was used to perform the sweep protein purification test on the modified membrane with better lysozyme adsorption efficiency, and the purification efficiency of the modified membrane was compared.

本試驗中選用市售的新鮮雞蛋白為進料溶液來進行掃流式實驗，藉以純化溶菌酶。上述表2中之星號★係為進行掃流式實驗所選擇之奈米纖維膜樣品，共選取2組：In this test, commercially available fresh chicken protein was used as a feed solution for sweeping experiments to purify lysozyme. The asterisk in Table 2 above is the sample of nanofiber membrane selected for the sweep experiment. A total of 2 groups are selected:

(1)-COCl+1%幾丁聚醣+染料(65.36 mg/g)；以及(1) -COCl + 1% chitosan + dye (65.36 mg / g);

(2)0.5%幾丁聚醣+溴乙酸(168.194 mg/g)。(2) 0.5% chitosan + bromoacetic acid (168.194 mg/g).

12.1　經改質之PAN奈米纖維膜(-COCl-幾丁聚醣-染料)之製備實例12.1 Preparation example of modified PAN nanofiber membrane (-COCl-chitosan-dye)

(a) 　配製5%(體積/體積)醋酸水溶液，再分別將1 g幾丁聚醣溶於100 ml 5%(體積/體積)醋酸水溶液中，攪拌24h，靜置24hr。 (a) A 5% (v/v) aqueous solution of acetic acid was prepared, and 1 g of chitosan was dissolved in 100 ml of a 5% (v/v) aqueous acetic acid solution, stirred for 24 hours, and allowed to stand for 24 hr.

(b) 　裁剪PAN奈米纖維膜為直徑約6.7 cm大小(面積約為35.24 cm2 、重量約為0.3 g)，先用四位數天枰秤其重量，以3 M NaOH溶液改質(85℃，0.5 hr)反應後以去離子水沖洗乾淨，再浸泡0.1 N HCl溶液靜置數分鐘完全反應後，再將奈米纖維膜加入5 ml之1 mol SOCl2 溶液反應60℃，0.5 hr以去離子水沖洗乾淨，再加入5 ml 1%(重量/體積)幾丁聚醣醋酸溶液放於80℃烘箱中反應3 hr以去離子水沖洗乾淨。 (b) Cutting the PAN nanofiber membrane to a diameter of about 6.7 cm (area of about 35.24 cm 2 and weighing about 0.3 g), first weighing it with a four-digit scorpion and modifying it with a 3 M NaOH solution (85 °C, 0.5 hr) After the reaction, rinse with deionized water, and then soak for 0.1 N HCl solution for a few minutes. After complete reaction, add the nanofiber membrane to 5 ml of 1 mol SOCl 2 solution and react at 60 ° C for 0.5 hr. Rinse with deionized water, add 5 ml of 1% (w/v) chitosan acetic acid solution and place in an oven at 80 °C for 3 hrs and rinse with deionized water.

(c) 　先配製10 ml cibacron blue F3GA(0.25 mg/ml)溶解於水之溶液，再加入2 ml 20%(重量/體積)NaCl溶液混合均勻後，將先前改質好的奈米纖維膜加入5 ml cibacron blue F3GA/NaCl溶液中反應60℃ 1 hr，之後再加入100 mg Na2 CO3 完全溶解後反應60℃ 3 hr，以去離子水沖洗乾淨備用。 (c) First prepare 10 ml of cibacron blue F3GA (0.25 mg/ml) dissolved in water, add 2 ml of 20% (w/w) NaCl solution and mix well, then add the previously modified nanofiber membrane. 5 ml cibacron blue F3GA/NaCl solution was reacted at 60 ° C for 1 hr, then completely dissolved by adding 100 mg of Na 2 CO 3 , then reacted at 60 ° C for 3 hr, rinsed with deionized water for use.

(d) 　配置100 ml新鮮雞蛋白以1:9的體積比例與pH 7之磷酸鹽緩衝溶液混合之溶液，使其在10,000rpm下於4℃離心30 min，收集上清液換一個離心瓶再離心一次，去除沉澱物。 (d) Dissolve 100 ml of fresh chicken protein in a 1:9 volume ratio with a pH 7 phosphate buffer solution, centrifuge at 10,000 rpm for 30 min at 4 ° C, collect the supernatant for a centrifuge bottle. Centrifuge once to remove the precipitate.

(e) 　將改質過後的奈米纖維膜，在掃流式過濾器之轉速為0 rpm下固定體積收集，以進行對雞蛋白的吸附。吸附過程100 ml新鮮雞蛋白/pH 7磷酸鹽緩衝溶液一開始1 ml收集10管，之後10 ml收集9管。洗滌過程100 ml pH 7磷酸鹽緩衝溶液10 ml收集10管。脫附過程100 ml 1 M NaCl/pH 7磷酸鹽緩衝溶液10ml收集10管。 (e) The modified nanofiber membrane was collected in a fixed volume at a rotational speed of the sweep filter of 0 rpm to carry out adsorption of chicken protein. Adsorption process 100 ml of fresh chicken protein/pH 7 phosphate buffer solution was collected in 10 ml from the beginning of 1 ml, and then 9 tubes were collected in 10 ml. Washing process 100 ml of 10 ml of pH 7 phosphate buffer solution was collected. Desorption process 10 tubes were collected in 10 ml of 100 ml 1 M NaCl/pH 7 phosphate buffer solution.

實驗中因幾丁聚醣為大分子，製備在奈米纖維膜上使之孔隙變小，故本實驗是以固定體積收集，使用壓力範圍在0~30psi。將氰基修飾為幾丁聚醣所帶有之一級胺並固定化反應性染料的奈米纖維膜在表2所示之批次吸附溶菌酶的量為65.36 mg/g，而在掃流式系統回收率為68.35%，流失率約為16%。整個吸附突破曲線程序，如圖14所示，進料體積約在80 ml為突破點。而且突破曲線趨勢較低，幾乎是完全吸附，溶菌酶吸附效率比雜蛋白佳。表4顯示在整個吸附過程中，溶菌酶的吸附率為90%；總量蛋白質的吸附率為14%；以1M NaCl溶液脫附率為57.69%，純度提升了53.52倍。In the experiment, the chitosan was a macromolecule, and the pores on the nanofiber membrane were made smaller. Therefore, the experiment was carried out in a fixed volume, and the pressure range was 0 to 30 psi. The amount of the lysozyme adsorbed by the cyano group modified to a monoamine of chitosan and immobilized with a reactive dye in the amount shown in Table 2 was 65.36 mg/g, while in the sweep type. The system recovery rate is 68.35% and the wastage rate is about 16%. The entire adsorption breakthrough curve program, as shown in Figure 14, has a feed volume of approximately 80 ml as a breakthrough point. Moreover, the trend of the breakthrough curve is relatively low, almost completely adsorbed, and the lysozyme adsorption efficiency is better than that of the hybrid protein. Table 4 shows that the adsorption rate of lysozyme was 90% in the whole adsorption process; the adsorption rate of total protein was 14%; the desorption rate was 17.69% in 1M NaCl solution, and the purity was increased by 53.52 times.

比活性(unit/mg)=活性(unit/ml)/蛋白質(mg/ml)Specific activity (unit/mg) = activity (unit/ml) / protein (mg / ml)

PFPF

(Fold)=254000/4754.54(Fold)=254000/4754.54

溶菌酶之產率(%)=沖提總活性(unit)/粗進料總活性(unit)x100Lysozyme yield (%) = total activity (unit) / crude feed total activity (unit) x 100

粗進料-膜分離-清洗=吸附(總活性(unit))Crude feed - membrane separation - washing = adsorption (total activity (unit))

回收率(%)=沖提/吸附(總活性(unit))x100Recovery rate (%) = extraction / adsorption (total activity (unit)) x 100

此外，將純化後所獲得溶液，利用鈉十二烷基的硫酸鹽(SDS)聚丙烯醯胺凝膠電泳(SDS-PAGE)來確認純化產物之分子量是否為溶菌酶(14.4 KD)，並且與市售之標準溶菌酶樣品做比較。Further, the solution obtained after purification was confirmed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) to determine whether the molecular weight of the purified product was lysozyme (14.4 KD), and Commercially available standard lysozyme samples are compared.

實驗結果如圖13所示，由1可知粗進料樣品具有許多雜蛋白與目標蛋白質(即溶菌酶)；由2(經膜分離後之樣品)可明顯看出本發明之奈米纖維膜吸附了目標蛋白質；由3(經清洗後之樣品)可知少量雜蛋白被洗出；以及藉由與5(溶菌酶標準品)相比，由4(經沖提之樣品)可知大量的目標蛋白質與極少量的雜蛋白被沖提出。The experimental results are shown in Fig. 13. It can be seen from 1 that the crude feed sample has many heteroproteins and the target protein (ie, lysozyme); from 2 (the sample separated by the membrane), the nanofiber membrane adsorption of the present invention is apparent. The target protein; a small amount of the heteroprotein was washed out by 3 (the washed sample); and a large amount of the target protein was known from 4 (the extracted sample) by comparison with 5 (lysozyme standard) A very small amount of the heteroprotein was flushed out.

12.2　改質PAN奈米纖維膜(-COCl-幾丁聚醣-溴乙酸)之製備實例12.2 Preparation Example of Modified PAN Nanofiber Membrane (-COCl-Chitosan-Bromoacetic Acid)

(a) 　配製5%醋酸水溶液，分別將0.5g幾丁聚醣溶於100 ml 5%(體積/體積)醋酸水溶液中。 (a) A 5% aqueous solution of acetic acid was prepared, and 0.5 g of chitosan was dissolved in 100 ml of a 5% (v/v) aqueous acetic acid solution, respectively.

(b) 　裁剪PAN奈米纖維膜為2cmx2cm大小，先用四位數天平稱其重量(約0.2 g)。以3 M NaOH溶液改質(85℃，0.5 hr)反應後以去離子水沖洗，再浸泡0.1 N HCl溶液靜置1分鐘反應後再以去離子水沖洗，再將改質奈米纖維膜(-COOH)加入50 ml之1 mol SOCl2 溶液反應60℃，0.5 hr後以去離子水沖洗，將改質完的奈米纖維膜個別加入5ml 1%(重量/體積)幾丁聚醣醋酸溶液反應80℃，3 hr。最後以去離子水沖洗乾淨。 (b) The PAN nanofiber membrane was cut to a size of 2 cm x 2 cm, and the weight (about 0.2 g) was first weighed with a four-digit balance. After upgrading with 3 M NaOH solution (85 ° C, 0.5 hr), rinse with deionized water, then soak for 0.1 N HCl solution for 1 minute, then rinse with deionized water, then modify the modified nanofiber membrane ( -COOH) Add 50 ml of 1 mol SOCl 2 solution to react at 60 ° C, 0.5 hr, rinse with deionized water, and add the modified nanofiber membrane to 5 ml of 1% (w/v) chitosan acetic acid solution. The reaction was carried out at 80 ° C for 3 hr. Rinse thoroughly with deionized water.

(c) 　將50ml 2 M NaOH溶液、50ml 1 M碳酸氫鈉溶液與250 mg溴乙酸均勻混合，以獲得一具有總體積為100 ml之溶液備用。 (c) 50 ml of 2 M NaOH solution, 50 ml of 1 M sodium hydrogen carbonate solution and 250 mg of bromoacetic acid were uniformly mixed to obtain a solution having a total volume of 100 ml.

(d) 　將自步驟(b)獲得之奈米纖維膜置於50 ml溴乙酸溶液中於60℃烘箱中反應3 hr後，以去離子水多次沖洗乾淨備用。 (d) The nanofiber membrane obtained from the step (b) was placed in a 50 ml bromoacetic acid solution in an oven at 60 ° C for 3 hr, and then rinsed with deionized water several times for use.

(e) 　配置100 ml新鮮雞蛋白以1:9的比例與pH 7磷酸鹽緩衝溶液混合之溶液，使其在10000rpm下於4℃離心30 min，收集上清液換一個離心瓶在離一次，去除沉澱物。 (e) Dissolve 100 ml of fresh chicken protein in a 1:9 ratio with a pH 7 phosphate buffer solution, centrifuge at 10,000 rpm for 30 min at 4 ° C, and collect the supernatant for one centrifuge. Remove the precipitate.

(f) 　將改質過後的奈米纖維膜，在掃流式過濾器之轉速為0 rpm下固定體積收集，進行對雞蛋白的吸附。吸附過程100 ml新鮮雞蛋白/pH 7磷酸鹽緩衝溶液一開始1 ml收集10管，之後10 ml收集9管。洗滌過程100 ml pH 7磷酸鹽緩衝溶液，10 ml收集10管。脫附過程100 ml 1 M NaCl/pH 7磷酸鹽緩衝溶液，10ml收集10管。 (f) The modified nanofiber membrane was collected in a fixed volume at a rotational speed of the sweep filter of 0 rpm to carry out adsorption of chicken protein. Adsorption process 100 ml of fresh chicken protein/pH 7 phosphate buffer solution was collected in 10 ml from the beginning of 1 ml, and then 9 tubes were collected in 10 ml. Washing process 100 ml pH 7 phosphate buffer solution, 10 ml collection 10 tubes. Desorption process 100 ml 1 M NaCl/pH 7 phosphate buffer solution, 10 tubes were collected in 10 ml.

結果顯示，在無轉速無壓力下，在計算每個純化步驟的產率後，整個回收溶菌酶的程序，回收率可高達100%左右，但突破曲線只有10ml，如圖16所示。對於奈米纖維膜來說，在吸附與洗滌過程中，流失率約為63%，溶菌酶的吸附率為37%；總量蛋白質的吸附率為18%：以1M NaCl溶液脫附率為39.12%，但純度提升了58.88倍，整個純化過程如表6所示。The results showed that, under no pressure and no pressure, after calculating the yield of each purification step, the recovery rate of the whole lysozyme recovery procedure was as high as about 100%, but the breakthrough curve was only 10 ml, as shown in FIG. For the nanofiber membrane, the loss rate is about 63% in the adsorption and washing process, and the lysozyme adsorption rate is 37%; the total protein adsorption rate is 18%: the desorption rate in the 1M NaCl solution is 39.12. %, but the purity was increased by 58.88 times, and the whole purification process is shown in Table 6.

比活性(unit/mg)=活性(unit/ml)/蛋白質(mg/ml)Specific activity (unit/mg) = activity (unit/ml) / protein (mg / ml)

PFPF

(Fold)=254000/4754.54(Fold)=254000/4754.54

溶菌酶之產率(%)=沖提總活性(unit)/粗進料總活性(unit)x100Lysozyme yield (%) = total activity (unit) / crude feed total activity (unit) x 100

粗進料-膜分離-清洗=吸附(總活性(unit))Crude feed - membrane separation - washing = adsorption (total activity (unit))

回收率(%)=沖提/吸附(總活性(unit))x100Recovery rate (%) = extraction / adsorption (total activity (unit)) x 100

將純化後所獲得溶液，利用SDS-PAGE來確認純化產物之分子量是否為溶菌酶(14.4 KD)，並且與市售之標準溶菌酶樣品做比較。實驗結果如圖15所示，1(粗進料樣品)顯示有許多雜蛋白與目標蛋白質(即溶菌酶)；2(經膜分離後之樣品)可明顯看出奈米纖維膜吸附的目標蛋白質；3(經清洗後之樣品)顯示洗下少量的雜蛋白；相較於5(溶菌酶標準品)，4(經沖提之樣品)顯示沖提出大量目標蛋白質與少量的雜蛋白。The solution obtained after purification was confirmed by SDS-PAGE to determine whether the molecular weight of the purified product was lysozyme (14.4 KD) and compared with a commercially available standard lysozyme sample. The experimental results are shown in Fig. 15. 1 (coarse feed sample) shows many heteroproteins and target protein (ie lysozyme); 2 (sample after membrane separation) can clearly see the target protein adsorbed by nanofiber membrane ; 3 (sample after washing) showed a small amount of mixed protein; compared to 5 (lysozyme standard), 4 (samples eluted) showed a large amount of target protein and a small amount of heteroprotein.

本技術領域中具有通常知識者將會認識到，在不脫離本發明的寬泛發明構思的情況下能夠對如上所述的具體實施例加以改變。因此可以理解，本發明不局限於所揭露的特別具體實施例，但希望覆蓋由所附申請專利範圍定義的在本發明的精神和範圍內的各種改進。It will be appreciated by those skilled in the art that the specific embodiments described above can be modified without departing from the broad inventive concept. It is understood that the invention is not to be limited to the specific embodiments disclosed, but the various modifications of the scope of the invention and the scope of the invention.

圖1顯示PAN奈米纖維膜以100%乙二胺直接在不同反應溫度及時間下改質後對於AO7吸附效能的比較Figure 1 shows the comparison of the adsorption efficiency of PAN nanofiber membranes with AO7 after 100% ethylenediamine directly modified at different reaction temperatures and times.

圖2顯示本發明直接以100%乙二胺改質之PAN奈米纖維膜與市售膜在pH4-12環境下對於BSA之吸附效能的比較Figure 2 shows the comparison of the adsorption performance of PAN nanofiber membranes modified with 100% ethylenediamine in the present invention with commercially available membranes for BSA in a pH of 4-12 environment.

圖3顯示以幾丁聚醣與幾丁聚醣/GPTMS錯合物改質之(-COOH)奈米纖維膜對AO7吸附效能之比較Figure 3 shows the adsorption performance of AO7 on (-COOH) nanofiber membrane modified with chitosan and chitosan/GPTMS complex.

圖4顯示以幾丁聚醣與幾丁聚醣/GPTMS錯合物改質之(-COCl)奈米纖維膜對AO7吸附效能之比較Figure 4 shows the adsorption efficiency of AO7 modified by chitosan and chitosan/GPTMS complex modified (-COCl) nanofiber membrane.

圖5顯示對於經幾丁聚醣、EDA或HAD改質之奈米纖維薄膜固定Cibacron blue F3GA的量Figure 5 shows the amount of Cibacron blue F3GA immobilized on a nanofiber film modified with chitosan, EDA or HAD.

圖6顯示具有反應性染料之薄膜經固定不同金屬後對BSA的吸附效能之比較Figure 6 shows the comparison of the adsorption efficiency of BSA on films with reactive dyes after fixing different metals.

圖7顯示具有反應性染料之薄膜經固定不同金屬後對溶菌酶的吸附效能之比較Figure 7 shows a comparison of the adsorption efficiencies of lysozyme after fixing different metals on a film with reactive dyes.

圖8顯示以GTAC改質之薄膜對AO7吸附效能之比較Figure 8 shows the comparison of the adsorption performance of GTAC modified by GTAC.

圖9顯示以GTAC改質之薄膜對BSA吸附效能之比較Figure 9 shows the comparison of adsorption performance of BSA modified by GTAC.

圖10顯示以溴乙酸改質之薄膜對溶菌酶吸附效能之比較Figure 10 shows the comparison of the adsorption efficiency of lysozyme by a membrane modified with bromoacetic acid.

圖11顯示以溴乙酸改質固定化金屬薄膜對BSA吸附效能之比較Figure 11 shows the comparison of the adsorption efficiency of BSA modified with bromoacetic acid modified metal film.

圖12顯示以溴乙酸改質固定化金屬薄膜對溶菌酶吸附效能之比較Figure 12 shows the comparison of lysozyme adsorption efficiency by immobilized metal film modified with bromoacetic acid.

圖13顯示改質PAN膜(-COCl-幾丁聚醣-染料)以掃流式方式分離溶菌酶之SDS-PAGE圖，其中使用15%SDS-PAGE、蛋白質濃度為10~20μg以及溶菌酶分子量為14.4KDa；以及其中M代表蛋白質標準品；1代表粗進料樣品；2代表經膜分離後之樣品；3代表經清洗後之樣品；4代表經沖提之樣品；以及5代表溶菌酶標準品(2mg/ml)。Figure 13 shows a SDS-PAGE image of a modified PAN membrane (-COCl-chitosan-dye) by sweeping separation of lysozyme using 15% SDS-PAGE, protein concentration of 10-20 μg, and lysozyme molecular weight. Is 14.4 KDa; and wherein M represents a protein standard; 1 represents a crude feed sample; 2 represents a sample after membrane separation; 3 represents a washed sample; 4 represents an extracted sample; and 5 represents a lysozyme standard Product (2mg/ml).

圖14顯示改質PAN膜(-COCl-幾丁聚醣-染料)掃流式突破曲線與純化程序。Figure 14 shows a modified PAN membrane (-COCl-chitosan-dye) sweep-through curve and purification procedure.

圖15顯示改質PAN膜(-COCl-幾丁聚醣-溴乙酸)以掃流式方式分離溶菌酶之SDS-PAGE圖，其中使用15%SDS-PAGE、蛋白質濃度為10~20μg以及溶菌酶分子量為14.4KDa；以及其中M代表蛋白質標準品；1代表粗進料樣品；2代表經膜分離後之樣品；3代表經清洗後之樣品；4代表經沖提之樣品；以及5代表溶菌酶標準品(2mg/ml)。Figure 15 shows a SDS-PAGE diagram of a modified PAN membrane (-COCl-chitosan-bromoacetic acid) by sweeping separation of lysozyme using 15% SDS-PAGE, protein concentration of 10-20 μg, and lysozyme The molecular weight is 14.4 KDa; and wherein M represents a protein standard; 1 represents a crude feed sample; 2 represents a sample after membrane separation; 3 represents a washed sample; 4 represents a washed sample; and 5 represents a lysozyme Standard product (2mg/ml).

圖16顯示改質PAN膜(-COCl-幾丁聚醣-溴乙酸)掃流式突破曲線與純化程序。Figure 16 shows a sweeping breakthrough curve and purification procedure for a modified PAN membrane (-COCl-chitosan-bromoacetic acid).

Claims (15)

一種奈米纖維膜，其具有一胺基，該胺基經反應性染料、縮水甘油三甲基氯化銨(GTAC)或溴乙酸(bromoacetic acid)改質；該反應性染料係使用Cibacron Blue F3GA、Procion red HE3B、Green A dye、Blue Dextran中任一者；該具有胺基之化合物為以下結構式(A)~(F)中任一者： A nanofiber membrane having an amine group modified with a reactive dye, glycidyltrimethylammonium chloride (GTAC) or bromoacetic acid; the reactive dye is Cibacron Blue F3GA Any one of Procion red HE3B, Green A dye, and Blue Dextran; the compound having an amine group is any one of the following structural formulae (A) to (F): 如申請專利範圍第1項之奈米纖維膜，其中該奈米纖維膜係由一具有胺基之聚合物所構成。 The nanofiber membrane of claim 1, wherein the nanofiber membrane is composed of a polymer having an amine group. 如申請專利範圍第1項之奈米纖維膜，該反應性染料係Cibacron Blue F3GA。 Such as the nanofiber membrane of claim 1 of the patent scope, the reactive dye is Cibacron Blue F3GA. 如申請專利範圍第1項之奈米纖維膜，其中該奈米纖維膜可進一步藉由離子性鍵結或親和性作用結合金屬離子。 The nanofiber membrane of claim 1, wherein the nanofiber membrane further binds metal ions by ionic bonding or affinity. 一種製造如申請專利範圍第1至4項中任一項之奈米纖維膜之方法，其包含步驟：(1)將一具有氰基之奈米纖維膜直接與二胺化合物於30至90℃溫度下反應0.5至3小時反應；或(2)將一具有氰基之奈米纖維膜之氰基以鹼化水解處理部分地或全部地改質為羧基；將該具有羧基之奈米纖維膜與氯化硫醯基於30至90℃溫度下反應0.5至3小時，以改質羧基為醯氯基；以及將該具有醯氯基之奈米纖維膜與該二胺化合物於30至90℃溫度下反應0.5至3小時；或(3)將一具有氰基之奈米纖維膜之氰基以鹼化水解處理部分地或全部地改質為羧基後，再將該具有羧基之奈米纖維膜與幾丁聚醣於30至90℃溫度下反應0.5至3小時；或(4)將一具有氰基之奈米纖維膜之氰基以鹼化水解處理部分地或全部地改質為羧基；將該具有羧基之奈米纖維膜與氯化硫醯基於30至90℃溫度下反應0.5至3小時，以改質羧基為醯氯基；以及將該具有醯氯基之奈米纖維膜與幾丁聚醣於30至90℃溫度下反應0.5至3小時；以及 (5)將步驟(1)至(4)中所得任一具有胺基之奈米纖維膜與一反應性染料、GTAC或溴乙酸於25至100℃溫度下反應0.1至24小時；其中具有氰基之奈米纖維膜係由聚丙烯腈(PAN)或其共聚物所構成。 A method for producing a nanofiber membrane according to any one of claims 1 to 4, which comprises the steps of: (1) directing a nanofiber membrane having a cyano group directly with a diamine compound at 30 to 90 ° C The reaction is carried out at a temperature of 0.5 to 3 hours; or (2) the cyano group of the nanofiber membrane having a cyano group is partially or completely modified into a carboxyl group by alkali hydrolysis treatment; the nanofiber membrane having a carboxyl group is used. The reaction with ruthenium chloride is carried out at a temperature of 30 to 90 ° C for 0.5 to 3 hours to modify the carboxyl group to be a chloro group; and the nanofiber membrane having a ruthenium chloride group and the diamine compound are at a temperature of 30 to 90 ° C The reaction is carried out for 0.5 to 3 hours; or (3) the cyano group of the nanofiber membrane having a cyano group is partially or completely modified into a carboxyl group by alkali hydrolysis treatment, and then the nanofiber membrane having a carboxyl group is further used. Reacting with chitosan at a temperature of 30 to 90 ° C for 0.5 to 3 hours; or (4) partially or completely modifying a cyano group of a nanofiber membrane having a cyano group to a carboxyl group by alkali hydrolysis treatment; The carboxylated nanofiber membrane is reacted with lanthanum chloride sulfonate at a temperature of 30 to 90 ° C for 0.5 to 3 hours to modify the carboxylic acid. As acyl chloride group; and the nanofiber membrane having the acyl chloride group and the chitosan reacted at temperatures of 30 to 90 ℃ 0.5 to. 3 hours; and (5) reacting any of the nanofiber membranes having an amine group obtained in the steps (1) to (4) with a reactive dye, GTAC or bromoacetic acid at a temperature of 25 to 100 ° C for 0.1 to 24 hours; The base nanofiber membrane system is composed of polyacrylonitrile (PAN) or a copolymer thereof. 如申請專利範圍第5項之方法，其中該具有氰基之奈米纖維膜係由具有氰基之聚合物所構成，其中該聚合物係為聚丙烯腈或其共聚物。 The method of claim 5, wherein the nanofiber membrane having a cyano group is composed of a polymer having a cyano group, wherein the polymer is polyacrylonitrile or a copolymer thereof. 如申請專利範圍第5項之方法，其中該二胺化合物係為乙二胺或己二胺以及該反應性染料係Cibacron Blue F3GA。 The method of claim 5, wherein the diamine compound is ethylenediamine or hexamethylenediamine and the reactive dye is Cibacron Blue F3GA. 如申請專利範圍第5項之方法，其中該奈米纖維膜可進一步藉由離子性鍵結或親和性作用結合金屬離子。 The method of claim 5, wherein the nanofiber membrane further binds the metal ion by ionic bonding or affinity. 如申請專利範圍第5項之方法，其中該鹼化水解處理係將該具有氰基之奈米纖維膜於4至150℃溫度下0.1至30N的鹼溶液中鹼化水解1至600分鐘後，置於室溫下一0.1至10N的酸性溶液中1至1440分鐘。 The method of claim 5, wherein the alkalized hydrolysis treatment is carried out by alkalization of the cyano-nanofiber membrane in an alkali solution of 0.1 to 30 N at a temperature of 4 to 150 ° C for 1 to 600 minutes. It was placed in an acidic solution of 0.1 to 10 N at room temperature for 1 to 1440 minutes. 如申請專利範圍第9項之方法，其中該鹼溶液為NaOH溶液或KOH水溶液；以及該酸性溶液為HCl水溶液。 The method of claim 9, wherein the alkali solution is a NaOH solution or an aqueous KOH solution; and the acidic solution is an aqueous HCl solution. 如申請專利範圍第5項之方法，其中該反應性染料之濃度為0.05至10mg/ml；該GTAC之濃度為0.1至50M；該溴乙酸之濃度為0.001至1M；該二胺化合物之濃度為0.1M至50M；該氯化硫醯基之濃度為0.01至10M；以及該幾丁聚醣之濃度為0.01至3%(重量/體積)。 The method of claim 5, wherein the concentration of the reactive dye is 0.05 to 10 mg/ml; the concentration of the GTAC is 0.1 to 50 M; the concentration of the bromoacetic acid is 0.001 to 1 M; and the concentration of the diamine compound is 0.1M to 50M; the concentration of the thiocyanate group is 0.01 to 10M; and the concentration of the chitosan is 0.01 to 3% (weight/volume). 如申請專利範圍第5項之方法，其中在步驟(3)之該幾丁聚醣與該 奈米纖維膜之羧基反應以及步驟(4)之該幾丁聚醣與該奈米纖維膜之醯氯基反應中，可加入GPTMS且一起於25至100℃溫度下反應0.1至24小時。 The method of claim 5, wherein the chitosan in the step (3) and the method In the reaction of the carboxyl group of the nanofiber membrane and the reaction of the chitosan of the step (4) with the chlorohydrazine group of the nanofiber membrane, GPTMS may be added and reacted together at a temperature of 25 to 100 ° C for 0.1 to 24 hours. 如申請專利範圍第12項之方法，其中該幾丁聚醣之濃度為0.01至3%(重量/體積)以及該GPTMS之濃度為1至100%(體積/體積)。 The method of claim 12, wherein the chitosan has a concentration of 0.01 to 3% (weight/volume) and the GPTMS has a concentration of 1 to 100% (vol/vol). 一種如申請專利範圍第1至4項中任一項之奈米纖維膜之用途，其係用於廢水處理、過濾細菌或蛋白質純化。 A use of a nanofiber membrane according to any one of claims 1 to 4 for use in wastewater treatment, filtration of bacteria or protein purification. 如申請專利範圍第14項之用途，其中該廢水處理係包括處理廢水中之重金屬和有機物質。 The use of the scope of claim 14 wherein the wastewater treatment comprises treating heavy metals and organic matter in the wastewater.
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