Preparation And Characterization Of Molecularly Imitated Organic Phosphorus Hydrolysis Mimic Enzymes

Since the glucose biosensor has been developed in 1962, the studies on biosensors based on enzyme as recognition elements are hot topics in biosensor development. Enzyme is widely used because of its high specificity and catalytic efficiency. Usually, the speed of biochemical reactions catalyzed by enzyme are ten to billions times faster than that of spontaneous reactions. However, the natural enzymes are not perfect. Up to now, most enzyme sensor devices or other protein-based biological molecule devices (such as protein-chip and protein computer, protein-based sensor, protein-based molecular machines, etc.) haven’t been widely used as expected. Aside from the problems resulted from equipment, other problems resulted from the enzyme itself, such as low stabilities, low life cycles and poor reproducibilities. These issues have a strong affect on the development of the bioanalytical devices for a long time.Biomimic enzymes, which are also known as artificial enzymes with enzyme-like catalysts to simulate the process catalyzed by enzymes, are synthesized by chemical methods. The molecular imprinting technique has been proved to one of the best ways to produce the binding sites of enzyme molecule. The polymers with enzyme-like activities, prepared by molecular imprinting, could overcome the shortcomings of the native enzyme, because they have good physical and chemical stabilities, long service life, well resistances to harsh environments, the simplicity of the preparation process and cheapness.Organophosphates are highly toxic compounds and can be used as chemical pesticides and nerve agents in wars and terrorist attacks. Therefore, the efficient degradation of organophosphates can not only reduce the pesticide residues, but also destroy the organophosphorus nerve agent. Organophosphorus hydrolase (OPH), also known as phosphotriesterase (PTEs), can widely hydrolysis organic phosphorus compounds, is a major enzymes to degradation organophosphorus agents. OPH not only can be used to deal with the degradation of organophosphorus pesticide residues and organophosphorus nerve agent, but also can be used to detect organophosphate biomimetic sensor. However, the content of organophosphorus hydrolase in natural and recombinant strains is too low, and environmentally sensitive, unstable, easy to deactivated, difficult to prepare. At present, OPH is not commercialized yet.In this paper, the biomimic OPH with high efficiency on catalytic hydrolysis organophosphorus was prepared by molecular imprinting technique, and the catalytic properties of the biomimic OPH were also studied.In chapter one, the current researchs on enzyme mimics, organophosphorus pesticides, organophosphorus hydrolase enzymes and preparation and application of molecular imprinting technique are the introduced, respectively.In chapter two, the commercialized 1-vinylimidazole and methacrylic acid were choosed as the functional monomer. diethyl(4-nitrobenzyl)phosphonate was used as template on transition state analog of paraoxon hydrolysis. The biomimic OPH was prepared by precipitation polymerization method under ultraviolet light irradiation. The dispersibility and shape of polymer particles is better, the particle size is uniform. When the ratio of template and functional monomers is 1:15, the biomimic OPH can effectively hydrolyze paraoxon. The molecularly imprinted polymer microspheres with OPH-like activity have high recognition and selectivity. Compared with the spontaneous hydrolysis of paraoxon, the imprinted polymer (MIP3) and non-imprinted polymer (NIP3) without template molecule conditions synthetic catalyzed hydrolysis of paraoxon activity were increased by 6.5×104 and 2.3×104 times, the catalyzed hydrolysis effectiveness of MIP3 increase by 2.8 times than NIP3, showing a good hydrolysis effect. Catalyzed hydrolysis of paraoxon maximum rate Vm is 0.48 mM/min, Michaelis constant Km is 0.11 mM.In chapter three, considering the structure of organophosphorus hydrolase, bifunctional monomer metal complexe was designed as functional monomer, while methyl methacrylate was used as assistant functions monomer, and a paraoxon hydrolysis transition state analog diethyl(4-nitrobenzyl)phosphonate was chosen as template. the biomimic OPH polymer microspheres was synthesized by precipitation polymerization method under ultraviolet light irradiation. Comparing with the spontaneous hydrolysis of paraoxon, the imprinted polymer (MIP) and non-imprinted polymer (NIP) without template molecule conditions synthetic catalyzed hydrolysis of paraoxon activity were increased by 1.6×105 and 7.9×104 times, showing a good hydrolysis catalysis efficeiency, which was 2.0 times than that of NIP. Catalysis hydrolysis of paraoxon maximum rate Vm is 0.38 mM/min, Michaelis constant Km is 0.025 mM. The artificial simulated OPH is expected to be used in both the degradation of organophosphorus pesticide residues and organophosphorus nerve agent, and the detection of organophosphate, so as to improve the practicability and the stability of the biosensor.