Synthesis, Characterization And Application Of Novel Hybrid Organic-inorganic Nanoflowers

Organic-inorganic hybrid nanomaterials, as a branch of nanosized materials, have attracted an increasing number of attentions over past few years. In particular, protein-phosphate hybrid nanoflowers have become a hot research topic in recent years, due to its unusual properties such as large specific surface area, hierarchical structure, and high enzymatic activity. In light of these advantages of the hybrid nanoflowers, a series of enzyme-inorganic hybrid nanoflowers were synthesized by a novel approach by using different enzymes and inorganic components, respectively. Significantly, the resultant enzyme-inorganic hybrid nanoflowers can be applied as immobilized enzyme reactor (IMERs) and colorimetric platform for highly efficient protein digestion and fast visual detection of small molecules. The contents of this paper include:In chapter 1, a general review of synthetic approaches and their research progress of the organic-inorganic hybrid nanomaterials were introduced, specially focusing on description of the classifications, synthetic methods, application and development of nanoflowers. In addition, the significance and content of this thesis were also briefly presented.In chapter 2, This study reports a facile approach for the synthesis of horseradish peroxidise (HRP)-inorganic hybrid nanoflowers by self-assembly of HRP and copper phosphate (Cu3(PO4)2·3H2O) in aqueous solution. Several reaction parameters that affect the formation of the hybrid nanoflowers were investigated and a hierarchical flower-like spherical structure with hundreds of nanopetals was obtained under the optimum synthetic conditions. The enzymatic activity of HRP embedded in hybrid naonflowers was evaluated based on the principle of HRP catalyzing the oxidation of o-phenylenediamine (OPD) in the presence of hydrogen peroxide (H2O2). The results showed that 506% enhancement of enzymatic activity in the hybrid nanoflowers could be achieved compared with the free HRP in solution. Taking advantages of the structural feature with catalytic property, a nano flower-based coloorimetric platform was newly designed and applied for fast and sensitive visual detection of H2O2 and phenol. The limits of detection (LODs) for H2O2 and phenol were as low as 0.5 μM and 1.0 uM by the naked-eye visualization, which meet well the requirements of detection of both analytes in clinical diagnosis and environmental water. The proposed method has been successfully applied to the analysis of low-level H2O2 in a spiked human serum and phenol in sewage, respectively. The recoveries for all the determinations were higher than 92.6%. In addition, the hybrid nano flowers exhibited excellent reusability and reproducibility in cycle analysis. These primary results demonstrate that the hybrid nanoflowers have a great potential for applications in biomedical and environmental chemistry.In chapter 3, the hybrid nanoflowers were synthesized in aqueous medium at room temperature using copper phosphate as the inorganic component and enzyme as the organic component. The effects of reaction parameters on the formation of the enzyme-embedded hybrid nanoflowers and its growth mechanism were investigated systematically. By monitoring the esterification reaction of N-a-benzoyl-L-arginine ethyl ester (BAEE), the enzymatic activity of trypsin in trypsin-dopped nano flower was calculated and the results showed 270% enhancement in enzymatic activity. The performance of such a microreactor was further demonstrated by digesting bovine serum albumin (BSA) and horseradish peroxidase (HRP), with a stringent threshod for unambiguous identification of these digests, the yielding sequence coverage for nanoflower-based digestion were 86% and 41%, the same as those obtained with the free enzyme. Whereas the digestion time of BSA or HRP in the former case was only 1 min, about 1/720 of that performed in the latter case (12 h). Furthermore, the nanoflowers exhibited an increased stability even after continuous use compared with that in free solution. In addition, the hybrid nanoflower-based IMER was applicable to the digestion of human serum, showing great promise for proteome research.In chapter 4, a hierarchical jonquil-like uniform hybrid nanoflower was synthesized, in which a-chymotrypsin acted as organic component and Ca3(PO4)2·nH2O as inorganic component. The morphology, constituent and pore size of the nanoflowers were characterized by scanning electron microscope (SEM), energy dispersive spectrometer (EDS), X-Ray Powder Diffraction (XRD), BET surface area measurement (BET), et al. The enzymatic activity of immobilized a-chymotrypsin was studied by monitoring the esterification reaction of N-Benzoyl-tyrosine ethyl ester (BTEE), and the results showed 266% enhancement under the optimal conditions. Moreover, the nanoflowers can also be used as IMER for high efficient BSA digestion, and the yielding sequence coverage for nanoflower-based digestion were 48%, higher than that obtained with the traditional in-solution enzymolysis. However, the digestion time of BSA in the former case was just 2 min, about 1/360 of that performed in the tranditional case (12 h). This indicated that the hybrid nanoflowers have great potential in proteomic analysis.