食品安全检测多通道酶纳米微阵列生物传感器芯片的构建研究

The food quality safety not only had caused the enormous risks to the public health, moreover seriously had also restricted the economy in China. It was essential to develop a rapid and sensitive screening method to ascertain the presence of hazardous compounds for food safety. It also had an important social significance and the actual demand. The literature material indicated that the enzyme biosensor had been widely applied in food safety inspection. However, the enzyme biosensor had poor stability, low precision, irreversible inhibition, single variety and other defects. The previous research proves that the novel acetylcholinesterase biosensor based on multilayer films containing nano-materials and acetylcholinesterase liposomes bioreactor exhibited good reproducibility and acceptable stability, thus putting forward hypotheses on the high efficiency biosensor based on various enzymes composites. In the present study, we describe the fabrication, characterization and analytical performance of four kinds of enzyme bioreactors based on spherical shells liposome by the film-evaporation combining with freeze-thawing method. The four-enzyme system (acetylcholinesterase, horseradish peroxidase, glucose oxidase and tyrosinase), immobilized on the transducer surface, was used as a bioselective element. Fabrication of multi-channel microchip biosensor based on microelectrode array of silicon dioxide cavities. Explore the hole and electron transport mechanisms within the multilayer film. Clarify the mechanism of the detection of many types of hazardous substances of the biosensor chip. A novel proposed idea was advanced for improvement of food safety inspection.

食品质量安全问题不仅对人类健康造成了极大危害，而且还严重制约了我国经济的发展。研发出适用于食品安全领域的高效快速成套的检测技术，有着重要的社会意义与实际需求。资料表明，酶生物传感器已开始应用于食品中有害物质的快速检测,但是，酶生物传感器却有着活性酶稳定性差，精确性低，不可逆抑制和检测品种单一等缺陷。前期研究发现，由乙酰胆碱酯酶生物反应器和不同纳米材料所构建的酶纳米可控多层膜，其稳定性和灵敏度得到较大改善。课题组据此提出复合有多种检测酶的高效酶纳米生物传感器的构建假设。本课题拟采用脂质体技术制备四种酶的生物反应器，筛选不同功能的纳米材料，采用微阵列技术构建多通道酶纳米生物传感芯片，探索多层纳米结构薄膜内空穴和电子的传输机制，阐明生物传感芯片的多种类物质检测机理，评估生物传感芯片的性能，明确检测酶的可再生方法，克服不可逆抑制，为食品质量安全检测技术的改良提供一种新的思路。

食品安全事件的频发，迫切需要构建一种低成本、高灵敏、快速可靠和能进行现场分析的检测技术。酶生物传感器技术因其具有分析速度快和易于微型化等优点而备受关注。其中，电化学型生物传感器因其能直接获取生物分子本身或反应体系的电子行为、易与电子技术结合开发小型分析检测系统等，所以已展现出了广阔的应用前景。资料表明，酶电化学生物传感器已开始应用于食品中有害物质的快速检测,但是，酶生物传感器却有着活性酶稳定性差，精确性低，不可逆抑制和检测品种单一等缺陷。本课题拟采用脂质体技术制备四种酶（乙酰胆碱酯酶、葡萄糖氧化酶、酪氨酸酶和辣根过氧化物酶）的生物反应器，筛选不同功能的纳米材料，采用微阵列技术构建多通道酶纳米生物传感芯片，探索多层纳米结构薄膜内空穴和电子的传输机制，阐明生物传感芯片的多种类物质检测机理，评估生物传感芯片的性能，明确检测酶的可再生方法，克服不可逆抑制，为食品质量安全检测技术的改良提供一种新的思路。主要的研究结果如下：.（1）采用薄膜蒸发-冻融法制备四种酶的脂质体，通过正交设计优化制备工艺，以鱼精蛋白沉淀法分离脂质体与游离酶。通过计算活性包封率，确定了四种酶的脂质体的最佳制备工艺。四种酶脂质体生物反应器相比于同等酶含量的游离酶，都具有较高的稳定性。.（2）采用硅酸钠为硅源，氯化铵为沉淀剂制备纳米二氧化硅。将多壁碳纳米管 (MWNTs)置于混酸中，利用超声波振荡截短碳纳米管、并使其与羧基链接。采用溶胶-凝胶法制备Al掺杂的ZnO纳米粉体。以AChE生物反应器（ALB）为模型，考察纳米材料的电化学特性。GCE-(ALB/SiO2)6和GCE-(ALB/MWNTs)6对农药的响应实验结果表明，农药抑制率与农药的浓度在一定范围内存在线性关系。GCE-(ALB/ZnO)5对展现了良好的光电性能，农药抑制率与农药的浓度在一定范围内存在线性关系。.（3）经过有序的组合构建酶膜，最终获得了以(ALB/MWNTs/ ZnO/SiO2)4膜为基础的性能优越的电化学生物传感器。采用模板法成功制备出了四种酶的微阵列电极，并对其进行了表征。结合微阵列和纳米多层膜研究结果，构建四种酶的传感芯片，并考察传感芯片的电化学响应特性。在实际样品的检测中，四种传感芯片均表现出良好的精确度和准确性，可以用于实际的检测，且不需要繁琐的样品预处理过程，能够满足快速检测的需要。