Immobilized Laccase By Using CoTAPC-Fe₃O₄ Nanoparticle Composite And Its Application On Fiber Optic Epinephrine Biosensor

Fiber optic sensor is one of the most important detection instruments. Fiber optic sensor has many advantages such as high sensitivity, quick response, easy operation and micromation. Fiber optic biosensor is an important branch of fiber optic sensor, and it has a wide application prospect in medical science, biological engineering, food industries and environment monitoring. Epinephrine is a crucial physiological neurotransmitter and is closely related to human health. As for the determination of epinephrine in the field of clinical diagnosis and biologic technology, a fiber optic biosensor is considered to be very necessary. The existing traditional methods for the determination of epinephrine have such deficiencies as low sensitivity or complicated operation that can't meet the need of practical use. Therefore, it's necessary to develop a novel fiber optic epinephrine biosensor.Biosensitive material is an important part of fiber optic biosensor. The characters of the material influence the whole fiber optic biosensor greatly. Organic-inorganic nanaparticle composite is a novel interdiscipline, it has good application in the future. Organic-inorganic nanaparticle composite has both the advantage of organic substance and inorganic nanocomposite. It has a wide application in immobilization of biological substances.In this thesis, a new kind of carrier cobalt tetraaminophthalocyanine (CoTAPc)-Fe₃O₄ with excellent characters was successfully prepared and was used in laccase immobilization. The immobilized laccase has good excellent performance. The fiber optic epinephrine sensor was designed and constructed, and several tests were performed to detect the concentration of epinephrine in solution.The work of this paper includes mainly four aspects as followed:(1) The CoTAPc-Fe₃O₄ nanoparticle composite was prepared by in situ complex technology . The composite were characterized by IR, FEG-SEM, XRD, EDS and Model 4HF VSM. The nanoparticle composites took the shape of roundish spheres with their mean diameter of about 70 nm. The nanoparticle composites have good dispersion, excellent compatibility and abundant active functions on the surface for immobilization of laccase.(2) The laccase was immobilized on the surface of the CoTAPc-Fe₃O₄ nanoparticle composite by crosslinking method. The effects of glutaraldehyde concentration, BSA concentration, the pH on the reaction process and the amount of laccase on the activity of immobilized laccase were studied. After immobilization, the best pH, temperature and Km of immobilized laccase catalyse ABTS were studied . The immobilized laccase has better stabilities in heat, storage and operation.(3) The reactive complexion of epinephrine's oxygenation in different pH were studied. When pH<7, the product was adrenochrome. When pH>7, the reaction was complicated. The product was adrenolutine at pH=9. This work provided a basis of epinephrine's detection in alkalescence.(4) A fiber optic epinephrine biosensor based on fluorescence quenching was designed and fabricated. The performance of this sensor in detecting epinephrine was investigated.