Study On Fiber Optic Biosensor For The Determination Of Adrenaline Based On The Catalysis Of Immobilized Laccase

Adrenaline can regulate the catabolism of liver, skeletal muscle and fat culatureto affect the catabolism of glucose and protein in human muscle. It also acceleratesthe decomposition of the hepatic glycogen, so as to enhance the reacting capacitywhen dealing with danger. Because adrenaline is so closely related to the humanhealth, their determination from blood, urine and other body fluids has an essentialrole in the diagnostics of some diseases. In normal situation, the concentration ofadrenaline in healthy subjects is quite low for urinary and plasma. Although sensitive,the existing traditional methods for the determination of adrenaline are relativelycumbersome and often need derivatisation and pre-concentration steps, which makethem not satisfactory for clinic diagnosis and pharmaceutics research. Therefore, it'svery necessary to develop a novel fiber optic adrenaline biosensor.The organic-inorganic nano composite is a novel multidisciplinarity researchfield. There has been considerable experimental and theoretical attention to thefunctionallization and potential applications of nano composite due to their uniquestructural, magnetic and photoelectric properties, which have become the researchfocus of material science. In this thesis, the organic-inorganic nano compositeimmobilized laccase was prepared and a fiber optic adrenaline bosensor based onfluorescence quenching was designed and fabricated. Finally, the principle and theperformance of the biosensor were investigated.The work of this thesis include the following aspects:(1) The ZnTAPc-Fe₃O₄ nanoparticle composite was prepared by in situ complextechnology. The composite were characterized by IR, FEG-SEM, XRD, EDS andhyssteresis loop. The nanoparticle composites took the shape of roundish sphereswith their mean diameter of about 15 nm. The nanoparticle composites have gooddispersion, excellent compatibility and abundant active functions on the surface forthe immobilization of laccase.(2) The pycnoporus sanguineus laccase was immobilized on the surface of the ZnTAPc-Fe₃O₄ nanoparticle composite by crosslinking method. The effects ofglutaraldehyde concentration, BSA concentration, the pH on the reaction process andthe amount of laccase on the activity of immobilized laccase were studied. WhenABTS was used as substrates, the immobilized laccases exhibited the maximumenzyme activity at pH 3.0 and 0℃. The Km value of the immobilized laccase was20.1μM, a little larger than that of the free laccase (12.6μM). The immobilizedlaccase has better stabilities in heat, storage and operation.(3) A fiber optic adrenaline biosensor based on fluorescence quenching wasdesigned and fabricated, using laccases immobilized on ZnTAPc-Fe₃O₄ composite ascatalyst, and ABTS was used as mediator. The mechanism of the change of phasedelay cure was studied. The detection range of adrenaline varied according to theactivity of the immobilized laccase and the concentrations of the mediator. Theperformance of the biosensor was investigated including analytical range (1.0×10^-5—2.0×10^-9M), response time (20-30s). The biosensor showed excellentreproducibility and stability and would be possibly used in real-time detection ofadrenaline in human urine and plasma in the near future.