Studies Of Characters Of Immobilized Laccase On Magnetic Chitosan Microspheres In Catalyzing Epinephrine

Fibre optic biosensor is a kind of fibre optic sensor. It has wide application in clinic and basic research because of its good compatibility, great flexibility. Adrenalin is a kind of nerve substance of catecholamine, which is an important parameter in human body and widely distributes in centrum nerve. At present, the methods to detect catecholamine include high voltage liquid chromatogram method, fluorescence method and so on. But all these methods operate complexly and can not meet the practical demand.Magnetic chitosan microsphere is a kind of organic-inorganic compound nanoparticle, which has magnetism and can be separated and oriented easily under applied magnetic field. Furthermore, the microspheres have small diameter and big face area.There are abundant reaction alive function group( — NH₂)on the surface ,which can immobilize substance with bio-activity such as immunity protein,enzyme. Therefore, bio-enzyme can get good catalyzed activity after their immobilization on the magnetic chitosan microspheres,which has important application in fiber optic bio-sensor based on enzyme catalyze.In this thesis, a new kind of carrier magnetic chitosan microspheres with excellent characters was successfully prepared and was used in laccase immobilization. A fiber optic epinephrine biosensor based on fluorescence quenching was designed and fabricated. Adrenalin in the water was detected through this device.The work of this paper includes mainly five aspects as followed:(1) The magnetic chitosan microspheres were prepared by applying reversed-phase suspension methodology. The microspheres were characterized by IR, FEG-SEM, XRD EDS and Model 4HF VSM. The microspheres took the shape of roundish spheres with their mean diameter of about 3 μ m. The microspheres have good dispersion, excellent consistent and abundant active functions on the surface for immobilization of laccase.(2) The laccase was immobilized on the surface of the magnetic chitosan microspheres by crosslinking method. The effects of glutaraldehyde concentration,the pH on the reaction process and the amount of laccase on the activity of immobilized laccase were studied.(3) The reactive complexion of epinephrine's oxygenation in different condition were studied.The result show that the optimal catalyzed temperature of the immobilized laccase was 55℃, the optimal pH for immobilized enzyme was 5.2. The immobilized laccase has better stabilities in heat,storage and operation . This work provided good basis for developing the fiber optic epinephrine sensor with excellent properties.(4) Using the cellulose acetate as matrix, the indicator is immobilized in CA membrane and a sensing membrane is formed.(5) A fiber optic epinephrine biosensor based on fluorescence quenching was designed and fabricated. The performance of this sensor in detecting epinephrine was investigated.