氧化石墨烯基质毛细管电色谱胰蛋白酶微反应器的性能研究
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摘要
基于石墨烯优良的物化性能,利用层层组装法将氧化石墨烯修饰于石英毛细管内壁,制备了氧化石墨烯基质的毛细管电色谱,通过电渗流、拉曼光谱等对其进行表征。在此基础上,基于离子键合法将胰蛋白酶固定于毛细管电色谱柱头,制备胰蛋白酶微反应器。两者结合构成毛细管电色谱胰蛋白酶微反应器。实验结果显示,氧化石墨烯作为基质既可提高样品的分离效率,还能促进胰蛋白酶的催化性能。氧化石墨烯修饰的毛细管电色谱对N-苯甲酰-L-精氨酸乙酯盐酸盐(BAEE)和N-苯甲酰-L-精氨酸(BA)混合物的分离度从裸毛细管的3.70提升至4.71,而其固定化酶活性(米氏常数K_m=1.10 mmol/L,最大反应速率V_(max)=0.32 mmol·L~(-1)·s~(-1))也明显优于裸毛细管(K_m=109.77 mmol/L,V_(max)=0.000 46 mmol·L~(-1)·s~(-1))。利用所制备的微反应器从10种中药材中筛选胰蛋白酶抑制剂活性成分的药材,结果发现三七和大黄中均存在胰蛋白酶抑制剂活性成分。
A capillary electrochromatographic column with graphene oxide(GO) as carrier was fabricated by a layer-by-layer assembly immobilizing the GO nanosheets onto capillary wall.The characterization on the colomn was carried out by measuring the electro osmotic flow(EOF) and Raman spectrum,etc.Based on this,an about 1 cm length of capillary electrochromatography trypsin micro-reactor was prepared by immobilizing the trypsin on end of capillary column through ion bonding.The performance of this technique was investigated.Results showed that the introduction of GO to the capillary column not only improved its separation efficiency on sample,but also promoted its enzyme catalytic performance.The separation degree of the GO-coated capillary on a mixture of N-benzuyl-L-arginine ethyl ester hydrochloride(BAEE) and N-benzuyl-L-arginine(BA) was increased from 3.70 to 4.71 compared with that of bare capillary.Meanwhile,the enzyme activity of immobilized enzyme with GO as carrier(Michaelis constant K_m=1.10 mmol/L,the maximum reaction rate V_(max)=0.32 mmol·L~(-1)·s~(-1)) was also enhanced significantly compared with that of the immobilized enzyme on unmodified capillary(K_m=109.77 mmol/L,V_(max)=0.000 46 mmol·L~(-1)·s~(-1).The prepared trypsin micro-reactor was finally employed as a model for the screening of enzyme inhibitors from ten kinds of herbs in the crude extracts.Results showed that the Pseudo-ginseng and the Rheum officinale both contained trypsin inhibitor activity,which indicated that the prepared enzyme immobilized micro-reactor is a potential tool for rapid inhibitor screening.
A capillary electrochromatographic column with graphene oxide(GO) as carrier was fabricated by a layer-by-layer assembly immobilizing the GO nanosheets onto capillary wall.The characterization on the colomn was carried out by measuring the electro osmotic flow(EOF) and Raman spectrum,etc.Based on this,an about 1 cm length of capillary electrochromatography trypsin micro-reactor was prepared by immobilizing the trypsin on end of capillary column through ion bonding.The performance of this technique was investigated.Results showed that the introduction of GO to the capillary column not only improved its separation efficiency on sample,but also promoted its enzyme catalytic performance.The separation degree of the GO-coated capillary on a mixture of N-benzuyl-L-arginine ethyl ester hydrochloride(BAEE) and N-benzuyl-L-arginine(BA) was increased from 3.70 to 4.71 compared with that of bare capillary.Meanwhile,the enzyme activity of immobilized enzyme with GO as carrier(Michaelis constant K_m=1.10 mmol/L,the maximum reaction rate V_(max)=0.32 mmol·L~(-1)·s~(-1)) was also enhanced significantly compared with that of the immobilized enzyme on unmodified capillary(K_m=109.77 mmol/L,V_(max)=0.000 46 mmol·L~(-1)·s~(-1).The prepared trypsin micro-reactor was finally employed as a model for the screening of enzyme inhibitors from ten kinds of herbs in the crude extracts.Results showed that the Pseudo-ginseng and the Rheum officinale both contained trypsin inhibitor activity,which indicated that the prepared enzyme immobilized micro-reactor is a potential tool for rapid inhibitor screening.
引文
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