Immobilized Laccase On Metaltetraaminophthalocyanine -Fe₃O₄ Nano Composites And Their Application In Fiber Optic Adrenaline Biosensor

The organic-inorganic nano composite is a novel material, formed by the nanoscale coplex of inorganic nanoparticles and the organic substance. It is an extremely promising research direction to apply this kind of materials to the field of life science and information technology. In this thesis, in order to solve the main problems existing in fiber optic sensors and clinical medicine detection, immobilized enzyme on organic-inorganic nano composites were prepared by using organic-inorganic nano complex technique and enzyme immobilization technique. The immobilized enzyme was successfully applied to oxygen-consumption optic fiber sensor based on enzyme catalysis. The detection of adrenaline, a very important physiological neurotransmitter in human body, has been realized.In this thesis, systematic studies have been done in the following aspects. Firstly, the organic-inorganic nano composites were prepared by in situ complex between metal tetraaminophthalocyanine (MTAPc, M=Cu, Co) and Fe304 nano particles, and the structure and properties of the MTAPc- Fe₃O₄ composites, especially those required as the support, were studied. Secondly, the catalyzed properties of the pycnoporus sanguineus laccase immobilized on MTAPc- Fe304 composites were investigated and compared with those of free laccase, and the optimum conditions of immobilization reaction were also achieved. Thirdly, the mechanisms of catalyzed oxidation of adrenaline in laccase system and laccase-ABTS [2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonat)] mediator system were studied, the optimum catalyzed oxidation conditions of the immobilized laccase were investigated. Fourthly, the optic oxygen-sensitive membrane was prepared and a fiber optic adrenaline biosensor was designed and constructed. Finally, the principle and the performance of the biosensor were investigated.The main conclusions of this thesis include the following five aspects:(1) The Fe₃O₄ nanoparticles with an average size of 10 nm were prepared by common chemical precipitation and two kinds of MTAPc (M=Cu, Co) were synthesized. The MTAPc-Fe304 composites were prepared by in situ complex and were characterized by FT-IR spectra, XRD, XPS, field emission gun SEM micrograph, and hysteresis loop. The results showed that MTAPc formed the covering layer on the surface of the composite. Several properties of MTAPc-Fe₃O₄ composites such as magnetic properties, thermal stability, antioxidation ability, solubility, and long-term stability have been studied. As the majority of the CuTAPc were attached to the surface of the composite, the Fe304 nanoparticles were shielded from oxygen and their stabilities was greatly improved. The magnetic properties, surface area, aperture and the stabilities of the MTAPc-Fe₃O₄ composites showed that the composites were good candidates as an enzyme support.(2) When ABTS was used as substrates, the pycnoporus sanguineus laccase exhibited maximum enzyme activity at pH 3.0, 550C and the Km value was 12.6/aM, which indicated this kind of laccase had a better catalyzed activity. The mechanism and the optimum reaction condition of catalyzed oxidation of adrenaline by pycnoporus sanguineus laccase were studied by cyclic voltammogram and UV-Vis spectroscopy and the final product of the oxidation reaction was adrenochrome. Laccase exhibited the maximal enzyme activity in 1.0 mol/L Na₂HPO₄-citric acid buffer solution at pH 5.0 and 50℃and the K_m value was 37.5μM. In laccase-ABTS mediator system, the mechanism showed that the catalyzed oxidation reaction of adrenaline consisted of four chemical equations, and the produced rate of adrenochrome depended on the rate constant of each equation and the experimental conditions such as the concentrations of laccase, substrate and mediator. UV-Vis spectroscopy showed that the produced rate of adrenochrome increased in the present of the mediator.(3) As CuTAPc-Fe₃O₄ composite and CoTAPc-Fe₃O₄ composite had no influence on the laccase catalysis reaction, the optimal immobilized conditions were the same for the two composites. The laccase was immobilized on the surface of the composite by crosslinking method and the whole immobilize process included four steps, The optimum pH, the optimum dosage, the optimum reaction time of activation of 100mg composite by glutaraldehyde were 6.0, 0.2g and 4h, respectively. The two optimum pH values, the optimum dosage for the BSA crosslinking reaction of 100mg composite were 8.0, 6.0 and 0.2g, respectively. The optimum pH, the optimum temperature and the optimum reaction time of immobilization of laccase were 5.0, 0℃C and 1h, respectively. When 2.0 mg/ml laccase solution was used, the immobilization yield and the activity of the immobilized laccase were 20%, 1430 U/g, respectively. When ABTS was used as substrates, laccases immobilized on CuTAPc-Fe₃O₄ composite and CoTAPc-Fe₃O₄ composite exhibited the same maximum enzyme activity at pH 3.0 and at 45℃. The Km value of the laccases immobilized on CuTAPc-Fe304 composite was 23.8/tM. When adrenaline was used as substrates, laccases immobilized on CuTAPc-Fe₃O₄ composite exhibited the maximum enzyme activity at pH 5.0 and at 55^C. In the laccase-ABTS mediator system, adrenaline was oxidized by immobilized laccases forming adrenochrome or adrenolutine.(4)Using the cellulose acetate (CA) as matrix, the optic oxygen-sensitive membrane was formed by embedding the indicator Ru(bpy)3C12 in the CA membrane. The optic oxygen-sensitive membrane was prepared with the content as follows: the solvent acetone is 3.5mL, H20 is 0.2mL, the indicator concentration is 8 mg/mL and the matrix is 0.1g. The calibration curve of optic oxygen-sensitive membrane showed that the concentration of the oxygen had a good linear relationship with the phase delay, the detection limit was 0.5mg/L, and the response time was less than 60s. The optic oxygen-sensitive membrane showed excellent reproducibility and stability and its working temperature was below 40℃.(5)A fiber optic adrenaline biosensor based on fluorescence quenching was designed and fabricated, using laccases immobilized on CuTAPc-Fe₃O₄ composite as catalyst, and ABTS was used as mediator. The mechanism of the change of phase delay cure was studied. The detection range of adrenaline varied according to the activity of the immobilized laccase and the concentrations of the mediator. The performance of the biosensor was investigated including analytical range (1.0×10^-5-2.0×10⁹M), response time (20-30s). The biosensor showed excellent reproducibility and stability and its working temperature was below 40℃.