Construction Of Nanomaterials Immobilized Enzyme Systems And Their Applications In Electrochemical Sensors

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.