Fe_3O_4-SiO_2纳米复合固定化葡萄糖氧化酶及应用
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摘要
光学生物敏感材料是光纤生物传感器中的关键组成部分。高性能的光学复合敏感材料的制备是研制高性能光纤生物传感器的关键技术。目前报道的基于酶催化的光纤生物传感器的敏感材料通常由光学敏感材料和生物酶两部分组成,如果将光学指示剂和酶通过合适的技术组合在一起制成光学复合敏感材料,可有效提高传感器的响应速度和检测精度,并可实现传感器的微型化,具有较高的应用价值。人体血液中葡萄糖浓度的检测对人体健康监测和疾病早期诊断十分重要,基于酶催化的光纤生物传感器是实现这类检测的有效手段之一,其研究具有重要的理论意义和应用价值。
本文制备Fe3O4-SiO2磁性纳米复合粒子(FSNPs),并以此为基体材料固定葡萄糖氧化酶(GOD),所得到的固定化酶具有优良的特性。制备了同时含有固定化酶和荧光指示剂的光学复合生物敏感材料,设计和构建了基于光学复合敏感材料催化的光纤葡萄糖传感器,初步研究了传感器的性能。
本论文的主要工作包括以下几个方面：
(1)采用共沉淀法制备具有超顺磁性的Fe3O4纳米粒子,粒径在15nm左右；通过溶胶-凝胶法制备形貌粒径单一的FSNPs。对所得的粒子利用透射电镜,振动样品磁强计进行表征。所得到的磁性纳米复合粒子平均粒径可达到60～80nm左右,接近超顺磁性,有良好的生物相容性。
(2)以FSNPs为载体通过交联法固定GOD,研究硅烷化试剂APTS浓度、戊二醛的浓度、最佳给酶量、酶固定化过程中的pH值等因素对固定化酶活性的影响；以葡萄糖为底物的固定化酶最佳使用温度为50℃,最佳使用pH值为7,Km值为34.9mM,与游离酶的相比增大。固定化酶具有较好的热稳定性、贮存稳定性和操作稳定性。
(3)制备了同时含有固定化酶和荧光指示剂的光学复合生物敏感材料,设计和构建光纤葡萄糖传感器测试系统,初步研究表明：传感器的检测范围为100～600mg/dl,响应时间为15s。
Biosensing material is an important part of fiber optic biosensor. The research on optical biosensing materials is the key technology to develop the fiber optic biosensors with good properties. The reported sensing materials for fiber optic biosensor based on enzyme catalysis usually consist of two parts, optical sensing part and catalysis part. If these two parts are combined into one using an appropriate method to form the opitcal complex sensing materials, the response rate and detecting precision of the sensor can be improved greatly, and the sensor micromation will probably be realized. This kind of work is very important to the application of fiber optic sensor based on enzyme catalysis. The detection of glucose concentration in blood is of great significance for human health monitoring and early diagnosis of diseases. Among the various detecting methods, fiber optic biosensor based on enzyme catalysis is an effective means. Therefore, the research on this kind of fiber optic biosensor is of great theory and application importance.
In this thesis, the magnetic Fe3O4-SiO2 nanoparticle composite (FSNPs) was successfully prepared and used as the carrier for glucose oxidase immobilization, and the immobilized glucose oxidase with good properties has been obtained. The optical complex biosensing materials containing both immobilized enzyme and fluorescent indicator were prepared. The fiber optic glucose sensor based on catalytic enzyme was designed and constructed, and the preliminary study on its properties has been performed.
The work of this paper includes mainly three parts as followed:
(1) Fe3O4 nanoparticle was prepared by co-precipitation method with size of 15nm. A good morphology and single size FSNPs was prepared by sol-gel method. The composite were characterized by TEM and Model 4HF VSM. The nanoparticle composite with an average size of 60-80nm is almost superparamagnetic and has promising biocompatibility.
(2) The glucose oxidase was immobilized on the surface of the FSNPs by crosslinking method. The effects of silane reagent APTS, glutaraldehyde concentration, the pH on crosslinking process and the amount of glucose oxidase on the activity of immobilized enzyme were investigated. After immobilization, the optimum pH, temperature and Km of immobilized enzyme catalyse glucose were 7,50℃and 34.9 respectively. The immobilized glucose oxidase has a better stabilities in thermal, storage and operation.
(3) The optical complex biosensing materials containing both immobilized enzyme and fluorescent indicator were prepared. The fiber optic glucose sensor test system was designed and fabricated, the preliminary research results show that its detecting range is 100～600 mg/dl and its response time is about 15s.
本文制备Fe3O4-SiO2磁性纳米复合粒子(FSNPs),并以此为基体材料固定葡萄糖氧化酶(GOD),所得到的固定化酶具有优良的特性。制备了同时含有固定化酶和荧光指示剂的光学复合生物敏感材料,设计和构建了基于光学复合敏感材料催化的光纤葡萄糖传感器,初步研究了传感器的性能。
本论文的主要工作包括以下几个方面：
(1)采用共沉淀法制备具有超顺磁性的Fe3O4纳米粒子,粒径在15nm左右；通过溶胶-凝胶法制备形貌粒径单一的FSNPs。对所得的粒子利用透射电镜,振动样品磁强计进行表征。所得到的磁性纳米复合粒子平均粒径可达到60～80nm左右,接近超顺磁性,有良好的生物相容性。
(2)以FSNPs为载体通过交联法固定GOD,研究硅烷化试剂APTS浓度、戊二醛的浓度、最佳给酶量、酶固定化过程中的pH值等因素对固定化酶活性的影响；以葡萄糖为底物的固定化酶最佳使用温度为50℃,最佳使用pH值为7,Km值为34.9mM,与游离酶的相比增大。固定化酶具有较好的热稳定性、贮存稳定性和操作稳定性。
(3)制备了同时含有固定化酶和荧光指示剂的光学复合生物敏感材料,设计和构建光纤葡萄糖传感器测试系统,初步研究表明：传感器的检测范围为100～600mg/dl,响应时间为15s。
Biosensing material is an important part of fiber optic biosensor. The research on optical biosensing materials is the key technology to develop the fiber optic biosensors with good properties. The reported sensing materials for fiber optic biosensor based on enzyme catalysis usually consist of two parts, optical sensing part and catalysis part. If these two parts are combined into one using an appropriate method to form the opitcal complex sensing materials, the response rate and detecting precision of the sensor can be improved greatly, and the sensor micromation will probably be realized. This kind of work is very important to the application of fiber optic sensor based on enzyme catalysis. The detection of glucose concentration in blood is of great significance for human health monitoring and early diagnosis of diseases. Among the various detecting methods, fiber optic biosensor based on enzyme catalysis is an effective means. Therefore, the research on this kind of fiber optic biosensor is of great theory and application importance.
In this thesis, the magnetic Fe3O4-SiO2 nanoparticle composite (FSNPs) was successfully prepared and used as the carrier for glucose oxidase immobilization, and the immobilized glucose oxidase with good properties has been obtained. The optical complex biosensing materials containing both immobilized enzyme and fluorescent indicator were prepared. The fiber optic glucose sensor based on catalytic enzyme was designed and constructed, and the preliminary study on its properties has been performed.
The work of this paper includes mainly three parts as followed:
(1) Fe3O4 nanoparticle was prepared by co-precipitation method with size of 15nm. A good morphology and single size FSNPs was prepared by sol-gel method. The composite were characterized by TEM and Model 4HF VSM. The nanoparticle composite with an average size of 60-80nm is almost superparamagnetic and has promising biocompatibility.
(2) The glucose oxidase was immobilized on the surface of the FSNPs by crosslinking method. The effects of silane reagent APTS, glutaraldehyde concentration, the pH on crosslinking process and the amount of glucose oxidase on the activity of immobilized enzyme were investigated. After immobilization, the optimum pH, temperature and Km of immobilized enzyme catalyse glucose were 7,50℃and 34.9 respectively. The immobilized glucose oxidase has a better stabilities in thermal, storage and operation.
(3) The optical complex biosensing materials containing both immobilized enzyme and fluorescent indicator were prepared. The fiber optic glucose sensor test system was designed and fabricated, the preliminary research results show that its detecting range is 100～600 mg/dl and its response time is about 15s.
引文
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[8]Xavier MP. Fiber-optic monitoring of carbamate pesticides using porous glass with covalently bound chlorophenol red[J]. Biosensors& bioelectronic,2000,14:895-905.
[9]Choi JW. Fiber optic biosensor for the detection of organophosphorus compounds using ACNE-immobilized viologen LB films[J]. Thin Solid Films,1998,327-329:676-680.
[10]杜伟杰,娄蕾,朱自强.改善酶固定以增强葡萄糖传感器的生物电化学活性[J].华东师范大学学报,2009(5)：37-44.
[11]张竹君,李建忠.用于人体血液中葡萄糖测定的光导纤维化学发光传感器的研究[J].陕西师大学报,1987(4)：57-61.
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