Combined physical and chemical immobilization of glucose oxidase in alginate microspheres improves stability of encapsulation and activity
Combined physical and chemical immobilization of glucose oxidase in alginate microspheres improves stability of encapsulation and activity
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DOI：
10.1021/bc050171z
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发表时间：
2005-11-01
影响因子：
4.7
通讯作者：
McShane, MJ
中科院分区：
文献类型：
--
作者：
Zhu, HG;Srivastava, R;McShane, MJ
利用固定化酶和蛋白质的化学传感器对于监测化学过程和生物系统是重要的。本研究采用乳化法制备钙交联海藻酸钠水凝胶微球作为酶的载体。采用三种不同的方法将葡萄糖氧化酶（GOx）包封在海藻酸微球中：物理包封（乳化）、化学缀合（缀合）以及物理包封和化学缀合的组合（乳化缀合）。利用层层自组装技术在海藻酸钠/葡萄糖氧化酶微球表面制备纳米有序涂层，以稳定生物环境下的水凝胶/酶体系。通过红外光谱分析、zeta电位分析和共聚焦激光扫描显微镜验证了海藻酸盐微球上GOx的包裹和纳米膜涂层的形成。为了比较酶包封技术的固定化特性和具有未涂覆微球的纳米膜的影响，在四周的时间内研究了酶负载、释放和有效GOx活性（每单位蛋白负载的酶活性）之间的关系。结果产生了四个关键发现：（1）与乳液技术相比，乳液缀合技术提高了海藻酸盐微球中GOx的稳定性，将GOx从微球中的浸出从50%减少到17%;（2）海藻酸盐纳米膜涂层随时间增加了GOx的稳定性，但也降低了有效GOx活性;（3）乳化结合技术的有效GOx活性（约3.5 × 10（-5）Au μ g（-1）s（-1））高于其他方法，四周内无显著变化;（4）当在一周后比较具有聚（烯丙基胺盐酸盐）/聚（苯乙烯磺酸钠）（{PAH/PSS}）涂层的三个双层的微球时，对于乳液缀合技术，GOx浓度最高。因此，这三种技术的比较表明，乳液共轭技术是一种潜在的有效和实用的方法来制备海藻酸盐/GOx微球植入式葡萄糖生物传感器的应用。
Chemical sensors utilizing immobilized enzymes and proteins are important for monitoring chemical processes and biological systems. In this study, calcium-cross-linked alginate hydrogel microspheres were fabricated as enzyme carriers by an emulsification technique. Glucose oxidase (GOx) was encapsulated in alginate microspheres using three different methods: physical entrapment (emulsion), chemical conjugation (conjugation), and a combination of physical entrapment and chemical conjugation (emulsion-conjugation). Nano-organized coatings were applied on alginate/GOx microspheres using the layer-by-layer self-assembly technique in order to stabilize the hydrogel/enzyme system under biological environment. The encapsulation of GOx and formation of nanofilm coating on alginate microspheres were verified with FTIR spectral analysis, zeta-potential analysis, and confocal laser scanning microscopy. To compare both the immobilization properties of enzyme encapsulation techniques and the influence of nanofilms with uncoated microspheres, the relationship between enzyme loading, release, and effective GOx activity (enzyme activity per unit protein loading) were studied over a period of four weeks. The results produced four key findings: (1) the emulsion-conjugation technique improved the stability of GOx in alginate microspheres compared to the emulsion technique, reducing the GOx leaching from microsphere from 50% to 17%; (2) the polyelectrolyte nanofilm coatings increased the GOx stability over time, but also reduced the effective GOx activity; (3) the effective GOx activity for the emulsion-conjugation technique (about 3.5 x 10(-5) AU mu g(-1) s(-1)) was higher than that for other methods, and did not change significantly over four weeks; and (4) the GOx concentration, when compared after one week for microspheres with three bilayers of poly(allylamine hydrochloride)/sodium poly(styrene sulfonate) ({PAH/PSS}) coating, was highest for the emulsion-conjugation technique. As a result, the comparison of these three techniques showed the emulsion-conjugation technique to be a potentially effective and practical way to fabricate alginate/GOx microspheres for implantable glucose biosensor application.