Preparation And Properties Of Sensitive Materials For Fiber Optic DCP Biosensor Based On Oxygen Consumption

2,4-chlorophenol (DCP) is a kind of very toxic substance among the pollutants in the environment and they are widespread, difficult to degrade resistant and toxic refractory. It had been regarded by United States Environmental Protection Agency(US EPA) and our government. as the toxic organic substance to be specially controlled. Therefore, the detection of DCP concerntion is of crucial importance to environmental protection and human health.The main goal of this thesis is to investigate the fiber optic DCP biosensors to detect DCP concentration. Immobilized laccase and copper phthalocyanine were used respectively as the bio-recognition element to constructed fiber optical DCP biosensors based on oxygen consumption, and the detection of DCP by fiber optic DCP biosensors was realized.In this thesis, the following aspects have been studied systematically. Firstly, the culture and purification conditions of Coriolus versicolor had been investigated. Laccase with the specific activity of 28.4 U/mg was prepared. Secondly, magnetic chitosan nanoparticles(MCN) were prepared by reversed-phase suspension method and used to immobilize laccase. The optimum conditions of immobilization were studied and the catalytic properties of free laccase and immobilized laccase were studied by using ABTS as the substrate. Thirdly, the mechanisms of the DCP oxidation catalyzed in laccase system and laccase-ABTS [2,2-azinobis -(3-ethylbenzthiazoline-6-sulfonat)] mediator system were studied, the optimum conditions for DCP oxidation catalyzed by free laccase and immobilized laccase were investigated. Fourthly, three types of metal phthalocyanine were synthesized by benzene bitter wine-urea Law, the mechanisms and the optimum conditions of DCP oxidation catalyzed by metal phthalocyanine were investigated. Finally, fiber optic DCP biosensors based on enzyme catalysis were designed and constructed. The principle and the performance of the biosensors based on DCP catalysed by laccase or copper phthalocyanine were investigated, respectively. The main conclusions of this thesis include the following five aspects:(1) By means of liquid fermentation, the culture conditions of Coriolus versicolor were optimized. The laccase was isolated with the specific activity of 28.4 U/mg. The catalytic properties of laccase were studied using ABTS as the substrate.The optimal conditions were determined as: potato juice 20%, glucose 20.0 g/L, peptone 4.0 g/L, CuSO₄ 1.0 mmol/L, KH₂PO₄ 3.0 g/L, MgSO₄ 1.5 g/L, ZnSO₄ 0.5 g/L, MnSO₄ 0.5 g/L, ABTS 1.0 mmol/L, Tween-80 0.10 %, pH 5.0, 130 rpm/min, 28℃,13 days.Laccase was isolated and purified through ultra-filtration, followed by ammonium sulphate fractionation, DEAE-Sepharose Fast Flow and Sephedax G-100 column chromatography. The specific activity of laccase was 28.4 U/mg and the molecular mass of laccase was estimated to be 64.4 kDa by SDS-PAGE. The molecular weight of isoenzymes was 35.8 kD.The catalytic properties of laccase were studied using ABTS as the substrate. The optimum pH value was 7.0 and the optimum temperature was 55℃. The K_m and V_max values of laccase on ABTS at 25℃were 7.78umol/L and 5.11×10^-5 L·mol ^-1·min^-1, respectively. The purified laccase represents excellent storage stability and could maintain 90.2% of its initial activity after stored at -18℃for 28 days.(2) MCN were prepared by reversed-phase suspension method and were characterized by TEM, NMR, FT-IR. The magnetic properties and oxidation resistance were investigated. The results showed that MCN were almost superparamagnetic with Hc of 10.27 Oe. The oxidation resistance of MCN was good. With small Hc, MCN wrer easy to be separated from solution and reused.The laccase was immobilized on the surface of MCN by crosslinking method and the process included two steps: MCN activation by glutaraldehyde and cross-linking by laccase. The activation process by glutaraldehyde was under the following conditions: 50 mg MCN were dipped in the PBS(pH 7.0) buffer for 12 hours, then activated with 1% glutaraldehyde for 2h at 25℃. After activation and treatment by magnetic separation method, the crosslinking reaction with laccase was carried out at 4℃. The optimum pH, laccase concentration and reaction time of crosslinking reaction were 6.0, 1.0 mg/mL and 4h, respectively. The immobilization yield and the activity of the immobilized laccase were 28 %, 670 U/g, respectively.The catalytic properties of immobilized laccase were investigated by using ABTS as the substrate. The immobilized laccases exhibited the maximum enzyme activity at pH 2.0 and at 50℃. The K_m and V_max values of immobilized laccase at 25℃were 23.38μmol/L and 3.53×10^-5 mol·L^-1·min^-1, respectively. Also, the immobilized laccases had good thermal, store, operation stabilities.(3) The mechanism of the DCP oxidation catalyzed by laccase and the optimum catalytic conditions by free laccase and immobilized laccase were studied. For both free laccase and immobilized laccase, the optimum pH was 8.0. The optimal temperature of free laccase and immobilized laccase were 45℃, 55℃, respectively. The kinetic study indicates that the DCP oxidation catalyzed by laccase coincided with first-order kinetic equation.In the laccase-ABTS mediator system, ABTS is employed to shuttle electrons from the DCP to laccase. It was shown that by using ABTS as a mediator, laccase from Coriolus versicolor was able to increase the rate of DCP oxidation.The mechanism of DCP oxidation catalyzed by laccase indicated that the catalyze process contained four reactions. The producing rate of 2-chloro-1,4-benzoquinon had relations with each reaction's rate constant, namely it was influenced by the concentration of laccase, mediator and substrate. For both free laccase and immobilized laccase in the laccase-ABTS mediator system, the optimum pH and temperature of DCP oxidiation catalyzed by laccase were 8.0, 50℃, respectively.(4) Three types of metal phthalocyanine were synthesized by benzene bitter wine-urea law. The mechanism and catalytic properties of DCP oxidation catalyzed by metal phthalocyanine were studied and the optimum catalytic conditions were as follows: pH 8.0, temperature 55℃, amount of metal phthalocyanin 5.0 mg/mL.(5) Two types of novel fiber optic DCP biosensors based on the DCP oxidation catalyzed by immobilized laccase or copper phthalocyanine were designed and fabricated. The performance of the biosensors was investigated. The linear detection range, detection limit and response time of the fiber optic DCP biosensor base on the catalysis of immobilized laccase were 1.0×10^-7-9.0×10^-5 mol/L, 4.4×10^-8 mol/L, 40 s respectively. The linear detection range, detection limit and response time of the biosensor base on the catalysis of copper phthalocyanine were 1.0×10^-6-9.0×10^-5 mol/L mol/L, 7.4×10^-7 mol/L, 600 s, respectively. Both of them showed good reproducibility and stability.Both of biosensors were used to detect the DCP concentration in the water from Yangtze river, and the results were compared those determined by HPLC, indicating that the detection of DCP concentration by these biosensors were reliable. The influences of interference ions Mg²⁺and Ca²⁺ on the detection of DCP concentration by these biosensors have been studied. For the fiber optic DCP biosensor base on immobilized laccase. The results were higher than the true values. The influences of Mg²⁺and Ca²⁺ were eliminated when EDTA was added. For the fiber optic DCP biosensor base on copper phthalocyanine, the result wouldn't be influenced by interference ions Mg²⁺and Ca²⁺.