摘要
光纤生物传感器是生物传感器的一个新的研究方向,与其它生物传感器相比,它具有检测灵敏度更高且不受电磁场干扰的优点,在近十年中取得的研究进展令人瞩目,已用于医学病原体、食物毒性、地下水污染、生化武器和环境样品等的快速检测。光纤葡萄糖生物传感器克服了传统分析方法的缺点,在众多生物传感器的研究中极为引人注目,对糖尿病的诊断有独到之处。
光学生物敏感材料是光纤生物传感器中一个非常重要的部分,其性能直接影响光纤生物传感器的性能。基于酶催化的光纤生物传感器是光纤生物传感器中的一大类,在很多领域具有重要的应用前景。有机—无机纳米复合材料由于其结构特殊、性能优异而成为材料学中的研究热点。有机—无机纳米复合粒子兼顾有机物和无机纳米粒子的诸多优点,且稳定性较好,以此为载体制备固定化酶,可作为用于光纤酶生物传感器的性能优良的敏感材料。
本文制备了四氨基苯基卟啉铜(CuTAPP)-Fe_3O_4纳米复合粒子,并以之固定了葡萄糖氧化酶(GOD),所得到的固定化GOD具有优良的特性。设计和构建了基于GOD催化的光纤葡萄糖生物传感器,对葡萄糖进行了初步的检测,检测到的荧光的滞后相移与葡萄糖溶液浓度存在较好的线性关系。
本论文的工作主要包括以下几个方面:
(1)利用原位复合法制备CuTAPP-Fe_3O_4纳米复合粒子。对所得的粒子利用红外、自振动样品磁强计、透射电镜等进行表征。所得到的纳米复合粒子的平均粒径可达到50 nm左右,接近超顺磁性,分散性好,有良好的生物相容性,而且表面有大量活性官能团(-NH_2),是一种优良的载体材料。
(2)以CuTAPP-Fe_3O_4纳米复合粒子为载体通过交联法固定了葡萄糖氧化酶(GOD),研究戊二醛的浓度、牛血清白蛋白的浓度、反应过程中的pH值、GOD的用量等因素对固定化GOD活性的影响,以葡萄糖为底物固定化GOD的最佳使用温度为40℃,最佳使用pH值为5.5。固定化GOD具有较好的热稳定性、贮存稳定性和操作稳定性。
(3)设计和构建了基于荧光猝灭原理的光纤葡萄糖传感检测系统,对葡萄糖进行了初步的检测,研究了其传感特性。
Fiber optic biosensor is a new research branch of biosensor.Compare with others,it has many advantages such as high detection and immunity from electromagnetism field.The research in fiber optic biosensor has get great progress in this ten years,and it has been widely used in medical science,biological engineering, food industries and environment monitoring.
Biosensitive material is an important part of fiber optic biosensor.The characters of the materials 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.Fiber optic glucose biosensor especially widly applied in detecting diabetes.
In this thesis,a new kind of carrier tetra(p-aminophenyl)porphyrin copper (CuTAPP)-Fe_3O_4 with excellent characters was successfully prepared and was used in GOD immobilization.The immobilized GOD has good excellent performance.The fiber optic glucose sensor was designed and constructed,and several tests were performed to detect the concentration of glucose in solution.
The work of this thesis includes mainly three aspects as followed:
(1)The CuTAPP-Fe_3O_4 nanoparticle composite was prepared by in situ complex technology.The composite were characterized by IR,TEM,XRD and EDS.
The nanoparticle composites took the shape of roundish spheres with their mean diameter of about 50 nm.The nanoparticle composites have good dispersion,excellent compatibility and abundant active functions on the surface for immobilization of GOD.
(2)The GOD was immobilized on the surface of the CuTAPP-Fe_3O_4 nanoparticle composite by crosslinking method.The effects of glutaraldehyde concentration, BSA concentration,the pH on the reaction process and the amount of enzyme on the activity of immobilized enzyme were studied.After immobilization,the best pH,temperature of immobilized GOD catalyse glucose were studied.The immobilized GOD has better stabilities in heat,storage and operation.
(3)A fiber optic glucose biosensor based on fluorescence quenching was designed and fabricated.The performance of this sensor in detecting glucose was investigated.
引文
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