【作者】 王周平；

【导师】 章竹君；

【作者基本信息】 西南师范大学 ， 分析化学， 2004， 博士

【摘要】 近年来，基于分离纯化酶的传统酶法分析日益受到新型生物催化活性材料如微生物、动植物组织、细胞、受体等，尤其是动植物组织的挑战，催化活性稳定、寿命长、取材容易、价格低廉等特性使得这类组织酶催化生物传感器／分析系统越来越受到人们的重视，相关研究成为酶法分析和生物传感器研究领域的一大热点。生物发光菌历来在食品安全、环境监测等方面发挥着重要作用，研究发现新型生物发光菌、构建新型无试剂型生物发光传感器是这一研究领域的主要任务之一。化学发光分析因其灵敏度高、线性范围宽、分析速度快、仪器设备简单便宜以及易于实现自动化和连续分析等特点，吸引着众多分析工作者的广泛关注，已被成功地应用于生物技术、药学、分子生物学、临床医学和环境检测等领域中许多重要的无机和有机物质的分析。 本研究工作主要集中于三个方面，一方面是将组织酶催化反应与光学检测系统(光度、荧光、化学发光)在线偶合，建立了一系列基于组织酶催化的流动注射光学分析系统；另一方面是发现了新型生物发光真菌-蜜环菌，对其气相和液相生物发光行为进行研究，初步构建了一种非常简单的无试剂型气体氧和溶解氧生物传感器；第三方面是建立新的化学发光分析方法，研究在线微透析、微超滤与其结合应用于药物-蛋白相互作用研究和将其应用于实际样品分析的可行性。 第一章为组织酶催化光学分析系统研究。1．1部分概述了组织酶催化分析研究的相Southwest Normal University， Doetor Thesis一Z.Wang(200126)关进展. 1 .2部分研究发现富含多酚氧化酶的蘑菇组织可催化氧化儿茶酚及儿茶酚胺类物质转化为有色的醒类和红类物质，这些产物的吸收峰较之原物质吸收峰明显红移。基于此，采用非常简单的固定化方法并结合流动注射技术，建立了一种简单、灵敏、准确、廉价的无试剂型儿‘茶酚、盐酸异丙肾上腺素、盐酸多巴胺流动注射紫外一可见分光光度分析系统。该系统对上述几种物质具有良好的响应，儿茶酚、盐酸异丙肾上腺素、盐酸多巴胺物质浓度在一定范围内与吸光度分别呈良好的线性关系(儿茶酚，2x10.6一l、10.3gmr’;盐酸异丙肾上腺素，4灰20一6一sxzo.‘9 ml‘’;盐酸多巴胺，4xlo“6一sxlo‘4gml一’)。对三种物质的检测限(3a)分别为:4x 10一，9 ml一’，1 .3xlo一‘gml一‘，z.oxlo一6gml‘’。将该系统用于药物制剂中盐酸异丙肾上腺素、盐酸多巴胺含量的测定，所得结果与药典标准方法测得结果一致。同时用蘑菇组织制作的反应器具有制作简单、高酶活、使用寿命长、易更换和成本低廉等优点。 1，3部分研究发现富含多酚氧化酶的蘑菇组织可替代纯多酚氧化酶催化氧化异丙肾上腺素使其转化为异丙肾上腺素红，该物质在碱性条件下可进一步发生重排而成为具有强烈荧光特性的三轻基叫垛类物质。基于此将蘑菇组织催化氧化异丙肾上腺素反应与流动注射荧光光度分析技术结合，建立了一种非常简单、灵敏和高选择性的异丙肾上腺素蘑菇组织催化氧化一流动注射荧光分析系统。在实验选定条件下，荧光强度与异丙肾上腺素浓度在 3xlo一8一1又10一，9 ml一’范围内呈良好的线性关系，检测限为1 .0x10一8 9 mrl(3。)，相对标准偏差小于5%(n二11)。将该系统初步应用于药物制剂中异丙肾上腺素含量的测定，所得结果与药典标准方法测得结果一致。采用流动注射技术同时解决了产物荧光不稳定、常规类似测定中需加入稳定剂等问题而无须考虑反应动力学因素。用蘑菇组织制作的反应器同样具有制作简单、高酶活、使用寿命长、易更换和成本低廉等优点。没有氧化剂的引入，也符合当前绿色化学的潮流。 1.4部分构建了一种基于萝卜组织催化的流动注射化学发光谷氨酞胺分析系统。萝卜组织富含谷氨.酞胺酶，可替代纯谷氨酞胺酶定量催化谷氨酞胺生成氨的反应，将这一催化反应与NBS一二氯荧光素化学发光体系测定氨的反应在线偶合，使催化反应与化学发光检测反应在不同位置发生，既保证了二者反应都在最佳条件下进行，又使得所制作的组织反应器寿命大大延长。在实验选定的最佳条件下，化学发光强度与谷氨酞胺浓度在7x10一8一l、10一，gmr’范围内呈良好的线性关系，检测限为2.3、10一sgml一，(3。)，相对标准偏差小于5%。将该系统应用于药物制剂、水样中谷氨酞胺含量的测定，结果令人满 2竺丝竺塑鱼竺些竺竺业丝竺竺卫些全圣丛些卫丝坐丝...一…~1「_…__……~_:煞少睽意。同时试验了将该系统用于生物体液中谷氨酸胺测定的可能性，标准加入实验表明该系统可成功用于血样中谷氨酞胺的检测，并具有监测谷氨酞胺类药物服用后血药浓度变化的潜能。 第二章为生物发光真菌.蜜环菌生物发光行为研究。2.1部分概述了生物发光的原理和相关研究进展。 2，2部分研究报道了一种天然生物发光真菌一蜜环菌的气相发光行为。结果显示在酸碱度、温度和培养基质适更多还原

【Abstract】 Recent years, traditional enzymatic analysis based on isolated and purified enzyme has increasingly underwent the challenge from the novel bioactive catalysis materials including microorganism, animal and plant organ or tissue, cell and receptor, especially from animal and plant tissue. The advantages of more stable catalytic activity, longer lifetime, easy for materials getting and low cost make the kind of tissue enzyme catalysis-based biosensor or analytical system more and more attractive. Research on it thereby has become a hotspot in the study field of enzymatic analysis and biosensor. Bioluminescent microorganisms have been playing an important role in the fields of food safe inspection, environmental monitoring and the others. The finding and study on novel bioluminescent microorganisms and construction of new reagentless bioluminescence biosensors is one of the main tasks for the researchers in the research field. Attributing to the excellent characteristics of high sensitivity, wide linear range, rapidity, simple instrument and suitable for automatization and continuous analysis, chemiluminescence have attracted many people’s attention and have been successfully applied for the determination of various important inorganic and organic compounds in the fields of biotechnology, pharmacology, molecule biology, clinic medicine and environmental monitoring.The present study focused on three aspects. The first is to on-line couple tissue enzymatic catalysis with spectrophotometric, fluorophotometric and chemiluminescent analysis systems and established serials of tissue enzymatic catalysis-based optical analytical systems. Thesecond involved in the finding and study on a novel and native bioluminescent fungi-Armillariaella mellea (AM). Based on the investigations on its bioluminescent behavior in gas and aqueous phase, a very simple and reagentless biosensor for gas oxygen and dissolved oxygen determination was developed. In the last aspect, some new chemiluminescence analytical systems and methods were proposed and the feasibility of coupling it to on-line microdialysis or microultrafiltration sampling for the study of drug-protein interaction and applying it for real sample analysis was examined.Chapter 1 Tissue enzymatic catalysis-based optical analytical systemsIn this chapter, correlative study advancement on tissue enzyme catalytic analysis was summarized briefly in Section 1.1 at first.In Section 1.2, a novel, simple, inexpensive and reagentless mushroom tissue-based spectrophotometric analytical system for the determination of catechol and catecholamines (isoprenaline and dopamine) combined with flow injection analysis is proposed. The mushroom tissue acts as the molecular recognition element. Catecholamines are oxidized by oxygen under the catalysis of polyphenol oxidase (PPO) in the tissue column to produce quinone. The concentrations of catecholamines are determined by monitoring the obtained products at their maximum absorption. The linear range of catechol concentration is 2xlO"6to 1 10-3 g ml-1 with the detection limit of 4 10-7 g ml-1 (3). The response to the concentration of isoprenaline is linear in the range of 4xlO"6~8*10"4 g ml"1 with a detection limit of 1.3x10"6 g mr’(3a). It is linear with the concentration of dopamine in the range 4xlO"6-8*10"4 g ml"1 with a detection limit of l.OxlO"6 g ml"1 (3a). The system has been successfully applied to the determination of isoprenaline and dopamine in pharmaceutical preparation. The obtained results are well in agreement with the values determined by the standard methods in pharmacopoeia.In Section 1.3, a novel simple and reagentless mushroom tissue-based fluorescence system for the determination of isoprenaline over the range of 3x10"8 -IxlO"5 g ml’1 combined with flow injection analysis is described. Mushroom tissue exhibits the rich activity of polyphenol oxidase (PPO), which was employed as molecular recognition elements. Isoprenaline was oxidized under the catalysis of PPO in tissue column to produce isoproteroquinone更多还原