Gustafsson Hanna, Küchler Andreas, Holmberg Krister, Walde Peter
Applied Surface Chemistry, Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden.
J Mater Chem B. 2015 Aug 14;3(30):6174-6184. doi: 10.1039/c5tb00543d. Epub 2015 Jul 7.
The two enzymes Aspergillus sp. glucose oxidase (GOD) and horseradish peroxidase (HRP) were co-immobilized on solid silica supports in a spatially controlled way by using mesoporous silica nanoparticles (Hiroshima Mesoporous Materials, HMM) and a polycationic dendronized polymer (denpol). The silica support was first coated with the denpol, followed by the deposition of the mesoporous silica nanoparticles into which - in a next step - GOD was adsorbed. Finally, the GOD-loaded silica nanoparticles were coated with a denpol-HRP conjugate constituting of several HRP molecules which were covalently bound to the denpol via bis-aryl hydrazone (BAH) bonds. The entire immobilization process was followed in real time with quartz crystal microbalance with dissipation monitoring (QCM-D). The activities and storage stabilities of the co-immobilized enzymes were determined by analyzing a two-step cascade reaction involving the two immobilized enzymes GOD and HRP. d-glucose and o-phenylenediamine (OPD) were used as substrates for GOD and HRP, respectively. The cascade reaction - in which intermediate hydrogen peroxide was formed from d-glucose and dissolved O with GOD - was shown to take place. The immobilized enzymes remained fairly stable for at least 2 weeks if stored in contact with an aqueous solution of pH = 7 at 4 °C. If, however, denpol-BAH-GOD coated HRP-loaded mesoporous silica nanoparticles were used (the reversed situation), the cascade reaction was not effective. This was probably due to slow diffusion of hydrogen peroxide from the surface-exposed GOD to the particle-trapped HRP, and/or due to an inefficient loading of active HRP inside the particles. Overall, the combination of two enzyme immobilization methodologies - enzymes adsorbed within mesoporous silica nanoparticles and enzymes adsorbed as denpol-BAH-enzyme conjugates - allows the spatially controlled localization of different types of enzymes in a simple way. Possible applications of the concept are in the field of bioelectrode fabrication.
通过使用介孔二氧化硅纳米颗粒（广岛介孔材料，HMM）和聚阳离子树枝状聚合物（denpol），将两种酶，即曲霉属葡萄糖氧化酶（GOD）和辣根过氧化物酶（HRP）以空间可控的方式共固定在固体二氧化硅载体上。首先用denpol涂覆二氧化硅载体，然后沉积介孔二氧化硅纳米颗粒，下一步将GOD吸附到其中。最后，用由几个HRP分子组成的denpol-HRP共轭物涂覆负载GOD的二氧化硅纳米颗粒，这些HRP分子通过双芳基腙（BAH）键与denpol共价结合。整个固定过程通过具有耗散监测的石英晶体微天平（QCM-D）进行实时跟踪。通过分析涉及两种固定化酶GOD和HRP的两步级联反应，测定了共固定化酶的活性和储存稳定性。d-葡萄糖和邻苯二胺（OPD）分别用作GOD和HRP的底物。结果表明，由d-葡萄糖和溶解的氧在GOD作用下形成中间产物过氧化氢的级联反应发生了。如果在4℃下与pH = 7的水溶液接触储存，固定化酶至少能保持相当稳定2周。然而，如果使用涂覆有denpol-BAH-GOD的负载HRP的介孔二氧化硅纳米颗粒（相反的情况），级联反应则无效。这可能是由于过氧化氢从表面暴露的GOD向颗粒捕获的HRP的扩散缓慢，和/或由于颗粒内部活性HRP的负载效率低下。总体而言，两种酶固定化方法的结合——酶吸附在介孔二氧化硅纳米颗粒内和酶作为denpol-BAH-酶共轭物吸附——以简单的方式实现了不同类型酶的空间可控定位。该概念的可能应用领域是生物电极制造。
