The immobilization of horseradish peroxidase on Ag/mesoporous silica nanocomposite

Abstract

The ordered mesoporous materials (OMMs) have been widely investigated for enzyme immobilization, and also growing in biosensor applications. With interesting feathers of OMMs, this thesis was aimed to investigate the influence of OMMs, as well as to develop OMMs to be the good carrier for enzyme immobilization and biosensor application. Therefore, the suitable OMMs and pH for immobilization were studied in the first part of this thesis. MCM-41, SBA-15, and MCF, were simultaneously investigated for the immobilization of horseradish peroxidase (HRP; E.C. 1.11.1.7). MCM-41 and SBA-15 were rod-like with respective average pore diameters of 32, and 54 Å, while that of MCF with spherical cell and frame structure was 148 Å. Moreover, these materials synthesized were of identical surface functional groups and similar contents of free silanol groups. At immobilization pH 6 and 8 almost 100% HRP loadings were obtained and in significant leaching were observed for all types of supports. However, MCF was found to give both the highest enzyme loading and leaching at pH 10. Maximum and minimum HRP activities were obtained at respectively immobilization pH 8, and 6. Activities of immobilized HRP increased with support pore diameters in the order: MCM-41 < SBA-15 < MCF. HRP immobilized at pH 8 gave the highest storage stability (both at 4℃ and room temperature), and in opposition to pH 6. In addition, HRP immobilized in MCF was found to be the most stable under storage. The finding should be useful for the creation of biocatalysts and biosensors. Therefore, MCF was further developed to aim as a biocarrier for biosensor application in this work. Silver nanoparticle was synthesized on MCF to enhance the electron transfer and it was expected to improve the properties of immobilized HRP. However, in the enzyme immobilization onto the pore of carrier, the mass transfer must be considered. Therefore, silica nanopowder was used in parallel to investigate mass transfer affecting HRP immobilization with MCF. For convenience, silica nanopowder was used as a model as a support of silver nanoparticles. Therefore, the synthesis conditions of Ag/SiO₂ nanocomposite by ultrasonication method were investigated in the second part of this thesis. Following the synthesis, the results showed that the adsorption time and silver precursor concentration significantly affected the number of silver nanoparticles. On the other hand, those synthesis parameters only slightly affected on the size of silver nanoparticles. Interestingly, the particle size and size distribution of the nanoparticles clearly showed that they could be controlled by the reduction time. Moreover, the functionalization reagent, (3-aminopropyl) trialkoxysilane was also found to act as an electron donor for the silver precursor. Since the sizes of silver nanoparticles were speculated to affect enzyme immobilization and biosensor application as the aim of this work. The synthesis conditions which showed the distinct size of silver nanoparticles were further used to synthesize Ag/MCF. Therefore, Ag/MCF was carried out under varying reduction time of 2 and 8 h, and fixing the silver nitrate concentration of 2000 ppm and adsorption time of 12 h. Ag/MCF was achieved to synthesize in similar conditions as Ag/SiO₂. The average size of silver nanoparticles which were attached inside the pore of MCF of both reduction times was similar at 5 nm. For enzyme based application, the different MCF supports and different silica nanopowder support were used to immobilize HRP. It was found that, the large surface area of different MCF support play an important role in enzyme loading; almost 100% of HRP was immobilized. However, less activity of immobilized HRP was obtained, around 35% when compared to free enzyme. On the other hand, different silica nanopowder support provided the higher activity of immobilized HRP. The highest activity of immobilized HRP was obtained from MSPs; it was up to 98% when compared to free enzyme. It was found that the silver nanopartilces inhibited the activity of immobilized HRP. The enzyme leaching from both types of supports were still observed. For biosensor application, silver nanoparticles play an important role in electron transfer. Silver nanoparticles enhanced the currents of immobilized HRP at electrode surface modified with MCF and silica nanopowder from -2.9 μA to -3.4 μA, and -2.0 to -3.4 μA, respectively. This indicates that Ag/MCF and Ag/SiO₂ is suitable to further application or develop as the receptor of biosensor.