【作者】 张艳；

【导师】 郭守武；

【作者基本信息】 上海交通大学 ， 材料科学与工程， 2013， 博士

【摘要】 本文通过两种途径以克服酶的纯化、分离过程复杂，成本昂贵，长期稳定性差，与底物以及产物不易分离，无法重复利用等缺陷。一是通过酶固定化方法，改善酶的稳定性及重复利用性。为此，本文选用纳米氧化锌（ZnO）和化学还原氧化石墨烯（CRGO）为酶固定化载体材料，构建了两种新型的纳米材料固定化酶体系。研究了所构筑的固定化酶体系中纳米材料与酶的作用机理以及材料对固定化酶催化性质和物理化学性质的影响。同时，利用纳米材料固定化酶制备出了电化学生物传感器。另一途径是设计并制备生物酶模拟物。该论文的具体研究内容及主要结果如下：（1）不同形貌的ZnO纳米材料用于酶的固定化体系的构筑。通过调节水热法制备过程中溶剂甲醇与水的比例，可控合成出了不同形貌的ZnO纳米颗粒，包括纳米球、纳米片、纳米多枝杈等。利用3-氨基丙基三乙氧基硅烷（APTES）和正硅烷（TEOS）对ZnO纳米材料表面进行了氨基功能化修饰。以戊二醛为交联剂，成功地将辣根过氧化物酶（HRP）通过化学键合的方法固在于氨基化的纳米ZnO材料表面。同时，研究了ZnO纳米粒子形貌对HRP固载的影响，发现三种不同形貌纳米ZnO材料达到最大酶固载量时所用戊二醛的量不同；HRP固载量以及固定化酶动力学参数也因载体材料形貌的不同而有差别；纳米材料的形貌对酶的固载量以及固定化酶的动力学参数都有着重要的影响。（2）氧化石墨烯/化学还原氧化石墨烯固载酶体系构筑。系统研究了HRP以及草酸氧化酶（OxOx）与氧化石墨烯（GO）及化学还原氧化石墨烯（CRGO）的相互作用，发现通过物理吸附，HRP和草酸氧化酶（OxOx）可成功固载于GO或CRGO表面，HRP和OxOx的固载量随着GO的还原程度的增加而增大。同时发现CRGO的酶固载量与溶液pH值无关，但受溶液中盐离子浓度的影响较大，盐离子浓度越大，酶的固载量越大，表明疏水作用是酶与CRGO结合的主要作用力。与GO固定化酶相比，CRGO固定化酶具有较好好的催化活性和重复利用性。特别是OxOx，固定化后的酶活升高至游离酶的1.4倍。（3）基于CRGO固定化酶的电化学传感器。由于CRGO具有良好的电学性质和大的比表面积，CRGO固定化酶可作为电化学电极修饰材料以制备相应的电化学传感器。本文利用CRGO固定化OxOx酶修饰玻碳电极后，发现在电化学循环伏安曲线上可以明显观测到OxOx催化草酸分解的特征峰，且随着草酸浓度的增加，特征峰电流不断增大。当采用OxOx固载量为5mg/mg，CRGO的覆盖量为0.6μg制备所谓酶电极时，对0.01-1.0mM区间范围内的草酸分解都具有很好的线性响应，较高的灵敏性和低的检测下限。（4）基于石墨烯量子点的生物酶模拟物。由于其完整的二维平面骨架结构和较多的羧基，石墨烯量子点（GQDs）具有良好的模拟过氧化物酶的催化活性。利用GQDs边缘富含的羧基，本文将GQDs通过化学键键合的方法结合于Au电极表面。GQDs修饰后的Au电极保留了GQDs对H₂O₂的催化活性。以GQDs/Au电极为基础构建的H₂O₂电化学传感器具有较宽的检测线性范围，低的检测下限，良好的稳定性和重复利用性，已被用于检测活细胞释放的H₂O₂浓度。更多还原

【Abstract】 Enzymes show usually unique catalytic properties, and have been widely utilized inmedical, chemical and food industries. However, natural enzymes assume severaldisadvantages. For example, their preparation and purification are usually time-consumingand expensive. The free enzymes can be easily denatured by environmental changes, andcan be digested by proteases. They are lack of long-term stability under process conditions,and also have difficulties in recovery and recycling.In this thesis, to overcome these problems, the enzyme immobilization andenzyme-mimetic materials, mainly the graphene quantum dots （GQDs）, were studied.Using nanoscaled ZnO particles with different morphologies, graphene oxide （GO）, andchemically reduction graphene oxide （CRGO） as substrates, the conjugates of nanoscaledmaterials and enzymes were constructed. The catalytic activities, physical and chemicalproperties of immobilized enzymes, and the interactions between enzymes and thesubstrates were studied systematically. The electrochemical biosensor based immobilizedenzyme was fabricated as well. As an enzyme-mimetic system, graphene quantum dots（GQDs）were explored. It was found that the GQDs showed pronounced peroxidase likecatalytic property. Meanwhile, the enzyme free electrochemical sensor to H₂O₂basedGQDs were studied. The main results of the work are as follows:（1） Immobilization of horseradish peroxidase （HRP） on ZnO nanocrystals withdifferent morphologies. The ZnO nanocrystals with different morphologies weresynthesized through a hydrothermal procedure, and the control on the morphology of ZnO nanocrystals was achieved by varying the ratio of CH3OH to H2O, which were used assolvents in the hydrothermal reaction. The surface of as-prepared ZnO nanoparticles wasfunctionalized with amino groups using3-aminopropyltriethoxysilane and tetraethylorthosilicate. Horseradish peroxidase was immobilized on the as-modified ZnOnanostructures with glutaraldehyde as a crosslinker. It was demonstrated that themorphologies of ZnO nanocrystals affected severely the HRP loadings and the catalyticalactivities of the immobilized enzyme.（2） Immobilizations of HRP and oxalate oxidase （OxOx） on graphene oxide andchemically reduced graphene oxide （CRGO）. The interactions between HRP and OxOxwith GO and CRGO were studied systematically. It was illustrated that the enzymes HRPand OxOx could be immobilized easily on both GO and CRGO through physicaladsorptions. Significantly, as the reduction extent of CRGO increased, the enzyme loadinggot higher. The enzyme loading onto CRGO can be tenfold higher than that on GO, andmaximum enzyme loadings reached1.3and12mg/mg for HRP and OxOx on CRGO,respectively. The enzyme loadings on CRGO were insensitive to pH, but affected by ionicstrength. The higher ionic strength resulted higher loading. The results suggested thathydrophobic interaction is the driving force for enzyme immobilization on the CRGO. TheHRP and OxOx immobilized on CRGO also exhibit higher enzyme activities andreusability than those on the GO. The results demonstrate that CRGO should be moreproper for enzyme immobilizations.（3） Electrochemical sensors based on CRGO immobilized with OxOx. Due to theultra-large specific surface area and excellent electrical conductivity, the CRGOimmobilized with enzymes has been considered as an ideal material for electrodemodification. It was found that the glass carbon electrode （GCE） modified with CRGOimmobilized with OxOx showed typical electrochemical catalytic property to oxalic aciddecomposition, and unique redox peaks appeared in the CV or DPV curves. Withincreasing of the OxOx loading, the electrochemical catalytic activity and the sensitivity of electrode can be improved. As a electrochemical sensor, the GCE modified the CRGOimmobilized with OxOx showed a linear detection range, from0.01mM to1.0mM, and adetection limit8μM （based on the S/N=3） to oxalic acid.（4） Peroxidase like catalytic property of graphene quantum dots （GQDs）. Due to theunique aromatic basal plan structure, small lateral size, and abound surface carboxylicgroups, GQDs exhibit intrinsic peroxidase-like activity. In the work, GQDs were preparedthrough photo-Fenton reaction GO. Using the periphery carboxylic groups, theas-synthesized GQDs were chemically assembled on Au electrode surface. It wasdemonstrated that, as an enzyme free electrochemical sensor to detect the H₂O₂, theGQDs/Au electrode exhibits wide linear H₂O₂detection range, low detection limit, goodstability and fast response, which is better than or comparable to many electrodesimmobilized enzymes. The electrodes could have potential application in H₂O₂sensing inbiological system.更多还原