流动注射聚离子敏感膜电极在生物传感中的应用

	流动注射聚离子敏感膜电极在生物传感中的应用
	雷佳宏
学位类型	硕士
导师	秦伟研究员
	2014-05-26
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	海洋化学
关键词	聚离子敏感膜电极流动注射分析生物传感器计时电位法肝素大肠杆菌o157 三磷酸腺苷
其他摘要	离子选择性电极是一类利用相界面电位变化来指示待测离子活度的电化学传感器，广泛应用于环境监测、食品安全和工业分析等领域。离子选择性电极因具有体积小、能耗低、携带方便、操作简便等特点，特别适用于现场快速分析。 20 世纪 90 年代，聚离子敏感膜电极的发现使采用离子选择性电极技术能够对肝素、鱼精蛋白和蛋白酶等生物大分子进行电位检测，拓宽了离子选择性电极在生物传感中的应用。然而，聚离子敏感膜电极受其电位响应本质的限制，通常不能重复使用。近年来，人们开发了多种可逆化的聚离子传感模式，实现了聚离子敏感膜电极的重复使用。然而，这些可逆化的聚离子传感模式很少与自动化分析仪器相结合，这在一定程度上限制了它们的应用和推广。基于此，本论文将聚离子敏感膜电极与流动注射分析技术相结合，发展了基于恒电流调控的流动注射聚离子敏感膜电极的电位传感模式，并成功应用于对聚离子、致病菌和生物小分子的连续快速检测。具体研究内容如下： 1. 基于恒电流调控聚阳离子释放技术的流动注射电位型肝素传感器：将聚离子敏感膜电极与流动注射分析技术相结合，发展了流动注射电位型聚离子传感模式。经主离子（聚阳离子鱼精蛋白）活化的聚离子敏感膜电极，其敏感膜表面释放的聚阳离子鱼精蛋白能够与样品中的聚阴离子肝素发生较强的静电作用，导致电极膜表面的鱼精蛋白浓度降低。在此情况下，样品溶液中的 Na⁺ 能够与电极膜相中的鱼精蛋白聚阳离子发生离子交换作用，膜相中 Na⁺ 浓度的增加导致电极电位降低。在外加恒电流的作用下，电极内充液中的鱼精蛋白向敏感膜表面的迁移速率增加，电极表面消耗的鱼精蛋白能够得到较快的更新，使电极能够快速再生。经过膜组分和流动分析条件的优化，该系统能够对聚阴离子肝素进行连续、快速的检测，检测的线性范围为 0.1 ~ 2.0 U mL^-1，检出限可达 0.06 U mL^-1（3σ）。本方法具有灵敏度高、可逆性强的优点，能够实现连续检测，已成功应用于血液样品中肝素的检测。 2. 基于核酸适体识别的流动注射电位型大肠杆菌 O157 传感器：以大肠杆菌 O157 的核酸适体为识别分子，以流动注射聚离子敏感膜电极为换能器，开发了一种连续检测致病菌的电位型生物传感器。样品中的聚阴离子核酸适体能够与电极膜表面释放的聚阳离子鱼精蛋白发生静电作用并产生电位响应；当核酸适体与其靶物质大肠杆菌 O157 作用后，核酸适体能够黏附于大肠杆菌细胞表面，这种结合作用制约其了与鱼精蛋白之间的静电作用，导致电位响应信号降低，从而实现对大肠杆菌 O157 的检测。通过与流动注射分析仪器联用，在外加恒电流的作用下，该方法能够对大肠杆菌 O157 进行连续检测，对大肠杆菌 O157 检测的线性范围为 10¹ ~ 10⁴ cfu mL^-1，最低可检出浓度为 10 cfu mL^-1。该方法灵敏度高、检测时间短，适用于环境样品中致病菌的现场快速检测。 3. 基于核酸适体识别的流动注射电位型三磷酸腺苷传感器：以三磷酸腺苷的核酸适体为识别分子，以流动注射聚离子敏感膜电极为换能器，开发了一种可以对生物小分子物质进行连续检测的方法。核酸适体能够与生物小分子三磷酸腺苷发生特异性识别作用并形成稳定的 G 型结构，其空间构型的变化能够有效抑制核酸适体与鱼精蛋白之间的静电相互作用，并导致电极电位信号降低，从而能够对三磷酸腺苷进行检测。研究表明，电极对三磷酸腺苷检测的线性范围为 2.0 ~ 12.0 μM，检出限可达 1.4 μM（3σ）。该方法具有一定的通用性，通过改变核酸适体序列，能够对其他生物小分子进行检测。 ; Ion-selective electrodes (ISEs) are a type of electrochemical sensors using phase boundary potential to indicate the activities of analytes. They have been widely used for determination of ionic species in environmental monitoring, food safety and industrial analysis. ISEs are suitable for on-site analysis owing to their attractive features including small size, low-energy consumption, portability and ease of use. In the 1990s, the development of polyion-sensitive membrane electrodes made it possible to realize potentiometric determination of biological macromolecules, such as heparin, protamine and proteases, which broadens the application fields of ISEs significantly. However, the polyion-sensitive membrane electrodes are not reversible due to the nature of their potential responses. In recent years, several detection modes have been established to improve the reversibility of the polyion-sensitive membrane electrodes. However, these detection modes have not been widely applied in automatic or flow analysis. In this thesis, a flow injection sensing mode of polyion-sensitive membrane electrode has been proposed. With an external current applied, the polyion-sensitive membrane electrodes can be used for continuous determination of polyions, bacteria and small biomolecules. The contents are shown as follows: 1. Flow injection potentiometric biosensor for continuous determination of heparin based on current-controlled release of protamine. A polyion-sensitive membrane electrode sensing mode based on flow injection analysis has been proposed. The polycation protamine released at the membrane interface of the polyion-sensitive membrane electrode, can electrostatically interact with the polyanion heparin in the sample solution, which induces the ion exchange of protamine in the membrane phase with Na⁺ in the sample solution, thus causing the change in potential. An external anodic current is continuously applied across the polymeric membrane to generate ion fluxes of protamine from the inner filling solution to the sample solution, which could make the electrode membrane regenerated quickly after each measurement. Under optimized conditions, the electrode can be used for determination of heparin rapidly and continuously. A good linear relationship between the potential peak height and the concentration of heparin in the sample solution can be obtained in the range of 0.1 ~ 2.0 U mL^-1, and the detection limit is 0.06 U mL^-1(3σ). The electrode shows high sensitivity and good reversibility. The proposed potentiometric sensing system has been successfully applied to determination of heparin in blood samples. 2. Flow injection potentiometric aptasensor for determination of Escherichia coli O157. A potentiometric aptasensor for detection of Escherichia coli O157 (E. coli O157) has been developed. The aptamer, as a kind of polyanion, can be indirectly measured by the flow injection polyion-sensitive membrane electrode via the electrostatic interaction between aptamer in the sample solution and protamine released at the membrane/sample interface. The molecular recognition between E. coli O157 and its aptamer leads to attachment of the aptamer to the cell surface of E. coli O157, which effectively prevents the aptamer from electrostatically interacting with protamine at the membrane surface, thus decreasing the potential response. With an external current applied, the electrode can be used for continuously monitoring of E. coli O157. The proposed aptasensing method enables the determination of E. coli O157 at concentrations down to 10 cfu mL^-1 with a linear concentration range of 10¹ ~ 10⁴ cfu mL^-1.With a high sensitivity and a short detection time, the proposed method is suitable for in-field rapid measurements of pathogen in the environment. 3. Flow injection potentiometric aptasensor for determination of adenosine triphosphate. A potentiometric aptasensor for detection of adenosine triphosphate (ATP) has been developed. The aptamer selectively binding to target ATP is used for molecule recognition and the flow injection polyion-sensitive membrane electrode is used for potential detection. The ATP aptamer binds specifically to the target molecules via reaction incubation, which induces a change in the aptamer conformation from a random coil-like configuration to a folded structure. Such target binding-induced aptamer conformational change decreases the flexibility of the aptamer molecule and effectively prevents the aptamer from electrostatically interacting with the protamine domain,which could decrease the potential response detected by the flow injection polyion-sensitive membrane electrode. Experimental results show that the method exhibits a good linear relationship between the potential response and the concentration of ATP in the concentration range from 2.0 ~ 12.0 μM. The detection limit is calculated to be 1.4 μM (3σ).
语种	中文
文献类型	学位论文
条目标识符	http://ir.yic.ac.cn/handle/133337/6812
专题	中国科学院烟台海岸带研究所知识产出_学位论文
推荐引用方式 GB/T 7714	雷佳宏. 流动注射聚离子敏感膜电极在生物传感中的应用[D]. 北京. 中国科学院研究生院,2014.

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