新型生物分析技术在环境医学监测中的研究

发布时间：2018-07-05 09:32

本文选题：酶热传感器 + 牛奶分析　；参考：《华中科技大学》2012年博士论文

【摘要】：社会的文明进步,科技的创新发展,人们在公共卫生安全、生活环境条件等多方面有较大改观的同时亦存在诸如环境污染对人体健康的影响日益突出,食品安全事件时有发生、糖尿病、癌症患者增多的趋势等严重的公共卫生问题,威胁着人类的健康和生命。以上问题都需要快速、有效的检测方法。随着科学技术日新月异,多学科交叉日益紧密,涌现出许多新兴生物分析技术,酶热生物传感器和生物芯片是具有代表性生物分析技术,反之,这些新兴生物分析技术的研发促进了科学技术研究和实践应用的快速发展。本论文目的是开展新型生物分析技术-酶热生物传感器和蛋白芯片分析技术在环境医学监测中的研究,为保障和维护人类健康提供有力技术支撑。酶热生物传感器是利用固定酶作为分子识别元件特异性催化底物,产生的热焓,通过热敏电阻转化成电信号,信号大小与底物浓度成正比,酶热传感器采用双通道,一个反应通道,一个参比通道,可以消除非特异性热,连续监测牛奶、血液等生物样中的关键物质如尿素、葡萄糖、乳酸,无需样品前处理,绿色环保,较其它技术方法具有快捷,精确、特异性高的种种优点,愈来愈引起分析生物学家及生物学界的广泛关注。已成为生物分析领域的主角之一。蛋白芯片随着蛋白组学的需要而发展起来的,具有高通量,快速的特点,"XNA on GoldTM"芯片是一种新型生物芯片,通过生物素-亲和系统构建自组装单分子膜,以提供一个强大的,多功能的固定表面。表面的疏水膜可以消除蒸发的影响,从而保持生物分子暴露在固定表面的活性。链霉亲和连接层可以连接任何生物分子类包括：碳水化合物,多肽,抗体,受体,以及更传统的基于阵列的DNA。本课题应用酶热生物传感器分析牛奶质量控制关键指标尿素、乳酸,全血中葡萄糖、乳酸,这将促进我国生物样品系列检测技术的方法学研究及其实践应用,为修订、完善我国检测技术标准体系积累科学资料、提供技术支持、拓展科研空间：再则,基于酶联放大信号技术用酶热生传感器检测肺癌标记物表皮生长因子受体EGFR的核苷酸杂交热信号,"XNA on GoldTM"生物芯片技术分析蛋白标记物癌胚抗原CEA,在基因、蛋白质两个水平初步探索肺癌标记物的检测方法,取得了一些有意义结果。今后目标是建立多标记物高通量分析方法并进行病人样本分析,考察标记物在人群中的早期筛查价值,为多层次,多水平研究环境因素、肺癌早期诊断、流行病之间联系打下良好基础。本论文将分四个章分别描述酶热生物传感器和蛋白芯片技术在环境医学监测中的研究。第一章酶热传感器检测牛奶中尿素和乳酸目的：建立酶热传感技术检测快速、分别检测牛奶中尿素和乳酸的方法。方法：食品检测一般需要繁琐的样品前处理以消除基质干扰,一种双通道流动注射分析传感器可以无需样品前处理,仪器应用热传导器检测来自酶与底物产生的热量,仪器的一个通道用于反应酶柱,另外一个通道用于消除非特异性热。此仪器可以测量牛奶中尿素和乳酸,无需样品前处理。结果：在缓冲溶液中尿素线性范围0.1-50mM(R=0.996),乳酸线性范围0.025-5.0mM(R=0.9998)),脂肪含量为0.5%,1.5%,3,0%牛奶中尿素相关系数R分别为0.994,0.992,0.990,乳酸相关系数R均为0.999,样品分析时间为5min/样,无需样品前处理可以缩短分析时间、简化操作、减少人员培训,另外双通道可以提高测量重复性、灵敏度、稳定性。结论：本方法操作简单,反应迅速,能满足牛奶样品中尿素、乳酸的监测需要,将在卫生、农业、环境发挥巨大作用具有较好的社会价值和经济前景。第二章酶热传感器同时检测血中葡萄糖和乳酸目的：建立酶热传感技术同时检测全血中葡萄糖和乳酸的方法。方法：糖尿病人通过连续监测血糖消除危险因素,血糖水平被认为是健康重要的指标,但单一个指标不能给出完整的信息；乳酸是细胞在无氧代谢下的产物,能用来监测细胞内有氧代谢和无氧代谢的平衡,同时测量两个代谢物可以提高病人治疗效果。用分流双通道酶热传感器同时测量全血中葡萄糖和乳酸,葡萄糖氧化酶分解葡萄糖,乳酸氧化酶分解乳酸。结果：试验中全血无堵塞和干扰发生,葡萄糖和乳酸检测范围均为0.5-45.0mM,葡萄糖标准方程Y=2.11x+8.09(r2=0.999),乳酸标准Y=1.86x+1.60(r2=0.999),分析时间2.5min/样。与商业化仪器HemoCue比较,两者有较好相关系数,酶柱活性可以保持45天有效测量,线性范围和灵敏度能满足临床分析需求,无需样品处理能够实现临床病人实时在线监测分析全血样品。结论：该方法操作简单、反应快速,无需样品前处理,能满足日常临床病人血液中葡萄糖和乳酸的同时监测。第三章酶热传感器检测表皮生长因子受体杂交热信号初步研究目的：建立基于酶热生物传感技术测定肺癌标记物表皮生长因子受体核苷酸杂交的热信号,为肺癌突变早期筛查提供新的有效方法。方法：把单链核苷酸固相在玻璃微球CPG上作为探针,完全配对的生物素标记的核苷酸与探针杂交反应,链霉亲和素偶联的碱性磷酸酶(AP-strep)通过与生物素作用结合到固相表面,底物4-硝基苯磷酸二钠盐(pNPP)与酶反应可以产生热信号。结果：Tris buffer选为流动相和底物溶解液,流速为0.5mlmin-1,底物浓度选为10.0mgmL-1。结论：初步探讨核苷酸杂交热信号酶热生物传感器检测方法,为以后工作打下基础。第四章蛋白芯片检测癌胚抗原初步研究目的：建立双抗体夹心法蛋白芯片检测癌胚抗原的方法,为肺癌早期筛查提供有力技术支撑。方法：本实验应用BioDot芯片点样仪在"XNA on GoldTM"芯片上采用双抗体夹心法原理测定癌胚抗原(CEA)水平,用生物素标记的癌胚抗原(CEA)抗体包被芯片,制成固相抗体,往包被芯片中先后加入癌胚抗原(CEA),荧光cy3标记的癌胚抗原(CEA)检测抗体,形成抗体-抗原-抗体复合物,经激光共聚焦(CCD)扫描后,信号的大小和样品中的癌胚抗原(CEA)呈正相关。结果：筛选系列捕获抗体、检测抗体浓度,最佳浓度均为0.1mgmL-1,分析系列抗原浓度获得标准曲线。结论：初步探讨了蛋白芯片测定癌胚抗原方法,为以后建立多标记物高通量检测方法打下基础。
[Abstract]:With the rapid development of science and technology , the development of science and technology , there are serious public health problems such as environmental pollution to human health , serious public health problems such as the occurrence of environmental pollution on human health , the occurrence of food safety incidents , diabetes , the increasing trend of cancer patients and other serious public health problems , which threaten the health and life of human beings .

The purpose of this paper is to carry out the research of new biological analysis technology - enzyme thermal biosensor and protein chip analysis technology in environmental medical monitoring to provide powerful technical support for guaranteeing and maintaining human health .

The enzyme thermal biosensor is used as a molecular recognition element - specific catalytic substrate by using a fixed enzyme , and the generated heat enthalpy is converted into an electric signal by a thermistor , the size of the signal is directly proportional to the concentration of the substrate , and the enzyme thermal sensor can eliminate non - specific heat and continuously monitor key substances such as urea , glucose and lactic acid in biological samples such as urea , glucose and lactic acid , and has the advantages of rapid , accurate and high specificity in biological samples such as urea , glucose and lactic acid , and has become one of the leading characters in the field of biological analysis .

A novel biochip for detecting lung cancer marker is composed of carbohydrate , polypeptide , antibody , receptor and more traditional array - based DNA .

In this paper , four chapters are divided into four chapters to describe the research of enzyme thermal biosensor and protein chip technology in environmental medical monitoring .

In chapter one , the enzyme thermal sensor detects urea and lactic acid in milk

Objective : To establish a method for rapid and rapid detection of urea and lactic acid in milk .

In chapter 2 , the enzyme thermal sensor simultaneously detects both glucose and lactic acid in blood

Objective : To establish a method for simultaneous detection of glucose and lactic acid in whole blood by using enzyme thermal sensing technique .
The results showed that glucose and lactic acid , glucose oxidase and lactate oxidase in whole blood were measured by shunt dual - channel enzyme thermal sensor .

A preliminary study on the detection of hybrid heat signal of epidermal growth factor receptor by enzyme thermal sensor

Objective : To establish a new effective method for the detection of lung cancer marker epidermal growth factor receptor ( EGFR ) by enzyme - thermobiosensing technique . A new effective method was provided for early screening of lung cancer mutation . The method comprises the following steps : the single - stranded nucleotide solid phase is used as a probe in the glass microspheres , the biotin - labeled nucleotide is hybridized with the probe , and the alkaline phosphatase ( AP - strep ) coupled by streptavidin is combined with the probe to generate a thermal signal .

A preliminary study on the detection of carcinoembryonic antigen by protein chip

Objective : To establish a method for the detection of carcinoembryonic antigen by double antibody sandwich method , which provides a powerful technical support for early screening of lung cancer .
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R734.2;R12

【参考文献】