|Place of Conferral||北京|
|Keyword||离子选择性电极 生物传感器 离子通量 氧化铝纳米通道 聚离子|
|Other Abstract||聚合物膜离子选择性电极检测技术由于具有仪器简单、选择性好、分析速度快、检测成本低、易于实现微型化等优点，在医学诊断、环境监测、食品分析等领域具有广泛的应用前景。近年来，伴随着聚离子选择性电极的发现和低检出限聚合物膜离子选择性电极的发展，离子选择性电极检测技术引起了人们的极大关注。目前，离子选择性电极的发展主要集中在以下几个方面：(1)高选择性离子载体的合成；（2）电中性分子的检测；（3）电极的微型化和阵列化；（4）离子通量的调控等。其中，如何利用穿越聚合物敏感膜的离子通量快速、灵敏地检测目标分析物是当前生命医学和环境科学中既新颖又极具吸引力的热门研究课题之一。本论文对聚合物膜离子选择性电极的离子通量进行了系统研究，开发了一系列成本低、操作简单、灵敏度高的电位型生物传感器。本研究工作主要包括两个方面：利用聚合物膜相向溶液相的离子通量，发展了测定胰蛋白酶活性和聚离子浓度的生物传感器；利用纳米通道调控从溶液相向膜相的离子通量，发展了电位型免疫传感器。具体内容如下：1．基于电流调控底物释放的聚阳离子选择性电极检测胰蛋白酶。在离子选择性电极离子通量可控释放的研究中，人们的研究对象主要是低电荷的无机离子，而对于多电荷的聚离子的研究很少涉及。本章研究了电流驱动聚阳离子可控释放聚合物膜离子选择性电极生物传感检测技术，采用宏命令控制计时电流法和计时电位法模式转换，成功实现了溶液中胰蛋白酶活性的无试剂化检测。其检测原理为：施加电流精确控制鱼精蛋白阳离子在电极膜表面的释放量，试样中的胰蛋白酶催化水解电极膜表面释放的鱼精蛋白，促使膜相中的鱼精蛋白与样品溶液中的钠离子发生离子交换，产生电极电位的变化，据此测定溶液中胰蛋白酶的活性。通过施加等同开路电位的恒电位，使电极在较短时间内实现界面的更新，实现电极的循环使用。实验中我们研究了电流的脉冲幅度、脉冲宽度以及电极内充液中鱼精蛋白的浓度对电极响应性能的影响。在最佳条件下，采用经鱼精蛋白活化的聚阳离子选择性电极，实现了胰蛋白酶的连续、可逆检测，线性范围为0.5-5 U mL-1，检出限达0.3 U mL-1。该研究为进一步开发以蛋白水解酶为标记物的电化学免疫分析提供新思路。2．基于离子通量调控的聚阳离子选择性电极检测聚阴离子。目前，聚离子选择性电极已成功用于肝素、鱼精蛋白以及软骨素等多种生物大分子的检测。然而，全血样品中高亲脂性的离子（如SCN- 、Sal-等）会严重干扰聚离子的检测。本研究以常见的抗凝血药物肝素为研究对象，以二壬基萘磺酸为阳离子交换剂，发展了一种基于鱼精蛋白可控释放的聚阳离子选择性电极，实现了全血中肝素的快速、高灵敏检测。其检测原理为：试样中的肝素与鱼精蛋白特异性结合，使膜表面渗出的鱼精蛋白浓度降低，导致膜相中的鱼精蛋白与溶液相中的钠离子发生离子交换，从而增加膜相中钠离子的活度，产生电极电位变化。由于低电荷的干扰离子（如 SCN-,Sal-）与鱼精蛋白的静电作用较弱，不会引发此离子交换过程，因此不会产生电极电位变化。本文详细研究了亲脂性有机盐四(4-氯苯基)硼酸四(十二烷基)铵（ETH 500）对电极响应性能的影响。研究表明，ETH 500的加入能够提高电极对钠离子的选择性，进而提高电极的稳定性。在最佳条件下，电极对全血中肝素的检出限可达0.1 U mL-1，线性范围为0.4-2 U mL-1，血液中常见的亲脂性离子不干扰肝素的测定，该研究为进一步开发聚离子活化的聚合物膜离子选择性电极技术提供依据。3．基于离子通量调控的聚阴离子选择性电极检测鱼精蛋白。体外循环后精确控制鱼精蛋白用量对抗肝素，是预防鱼精蛋白中毒性反应的关键环节。然而，对于血液基体，一方面血液中存在高浓度的亲脂性离子（如血液中钾离子的浓度约为5 mM，血细胞中钾离子的浓度高达 150 mM)，另一方面传统的聚阳离子选择性电极膜相中含有阳离子位点，在这种情况下，钾离子很容易进入聚合物膜相而干扰鱼精蛋白阳离子的测定。基于上述鱼精蛋白活化的聚阳离子选择性电极技术成功运用于全血中肝素的检测，本章又开发了肝素活化的聚阴离子选择性电极技术，成功实现了鱼精蛋白聚阳离子的检测，高浓度的钾离子不干扰测定。在最佳条件下，该电极对鱼精蛋白聚阳离子的检出限可达0.1 μg mL-1,线性范围为0.5-10 μg mL-1。4．高灵敏高选择性电位型传感器检测肝素。在聚合物膜离子选择性电极的研究中，人工合成的有机化合物常被用作离子载体掺杂于有机膜相中，而生物大分子被用作识别元素掺杂于电极膜中的研究还很少涉及。本章以鱼精蛋白-肝素体系为模型，构建了以鱼精蛋白作为分子识别元素的聚合物膜离子选择性电极技术，成功实现了肝素的快速、高灵敏检测。其检测原理为：将鱼精蛋白掺杂的电极膜浸入试样中，试样中的肝素诱导敏感膜内表面的鱼精蛋白扩散到溶液中并与其形成复合物。根据鲍林的电中性原理，膜相中多余的氯离子伴随着鱼精蛋白一同进入溶液相，使膜相中氯离子浓度降低，导致电极电位下降。在最佳膜组成条件下，在0.01-0.4 U mL-1 浓度范围内，初始电位变化速率与浓度呈良好的线性关系，检出限可达0.005 U mL-1。血液中常见离子（如SCN- 、Sal-等）不干扰肝素的测定。该研究为将具有高识别能力的生物大分子用于聚合物膜离子选择性电极的离子载体提供新思路。5．氧化铝纳米通道的制备及其在生物传感中的应用。纳米通道技术作为生物纳米技术研究的重要内容和新的生长点，为生物组分的有效分离和检测提供了新的手段。氧化铝纳米通道具有尺寸可控、易于修饰等优点，在生物物质分离与检测等领域有广泛的应用。目前，氧化铝纳米通道检测技术在生物检测中的应用主要集中在以氧化铝纳米通道为载体，根据电流、电阻以及电容信号变化检测目标分析物，而根据电位信号的变化达到检测目的的研究还很少涉及。本章采用二次阳极氧化法制备了高度有序的氧化铝纳米通道，并以生物素-亲和素为模型，采用聚合物膜离子选择性电极传导电位信号，氧化铝纳米通道为识别载体，验证了该方法检测生物大分子的可行性。我们采用此种方法成功实现了亲和素的检测，检出限为0.05 μg mL-1。为进一步开发纳米通道调控离子通量的聚合物膜离子选择性电极提供实验基础。6．基于氧化铝纳米通道的电位型免疫传感器 |
为了进一步发展基于纳米通道的生物传感器，本章发展了基于氧化铝纳米通道的电位型免疫传感器。利用戊二醛交联法将羊抗人的IgG固定在氧化铝纳米通道内，抗原分子与固定在纳米通道表面的抗体分子结合，形成抗原-抗体复合物堵塞纳米通道，阻碍指示离子通过纳米通道到达电极膜界面，从而产生电极电位的变化。实验中我们采用聚合物膜离子选择性电极传导信号，借助宏命令控制计时电流法/计时电位法交替技术，监测离子通量的变化。由于纳米通道的功能化修饰很难在微观上实现均匀一致，因此所开发传感器的重现性、响应范围以及检出限，还需要进一步提高。; Polymeric membrane ion-selective electrodes (ISEs) have valuable application in bio-medicine, environmental monitor, food industry, and drug due to their attractive features including high sensitivity, simple instrument, rapid response and low cost. With the development of polyion sensitive electrodes and low-detection-limit ISEs, much attention has recently been paid to the application of ISEs. Nowadays, the development of ISEs has been focused on detection of neutral molecules, synthesis of highly sensitive and selective ionophores, study of multielectrodes and microelectrodes and modulation of ion fluxes. The applications of ion fluxes through ISE membrane to detect the meaningful analytes have been a novel, attractive and hot topic in the current bio-medical studies and environmental science. In this thesis, a series of novel electrochemical biosensors strategies based on ion fluxes have been developed for determination of various biomolecules with low cost, ease in operation and high sensitivity. This paper includes two parts: making use of outward ion fluxes through ISE membrane to provide controlled-release protamine for monitoring enzyme activity and polyions; making use of inward ion fluxes through ISE membrane for measuring avidin and antibody. The contents of this thesis are as follows:1. A novel potentiometric biosensor for determination of trypsin is described based on current-controlled reagent delivery. During the past decade, ion fluxes across ion-selective electrode membranes have been found analytically useful, and most current applications are based on the ion fluxes of small inorganic and organic anions. The use of ISEs with current-controlled release of polyion, such as protamine, for sensitive detection and quantification of protease has not been realized to date. In this thesis, a polymeric membrane protamine-sensitive electrode with dinonylnaphthalene sulfonate as cation-exchanger is used for in situ generation and detection of protamine. Diffusion of protamine across the polymeric membrane can be controlled precisely by applying an external current. The hydrolysis catalyzed with trypsin in sample solution decreases the concentration of free protamine released at the sample-membrane interface and facilitates the stripping of protamine out of the membrane surface via the ion-exchange process with sodium ions from the sample solution, thus decreasing the membrane potential, by which protease can be sensed potentiometrically. The influences of anodic current amplitude, current pulse duration and protamine concentration in the inner filling solution on the membrane potential response have been studied. Under optimum conditions, the proposed protamine-sensitive electrode is useful for continuous and reversible detection of trypsin over the concentration range of 0.5-5 U mL-1 with a detection limit of 0.3 U mL-1. This detection strategy provides a rapid and reagentless way for measurement of protease activities and offers great potential in the homogeneous immunoassays using proteases as labels.2. Polycation-sensitive membrane electrode for the detection of heparin based on the controlled release of protamine.Currently, the application of polymer membrane-based potentiometric polyion sensors has evolved to provide a promising measurement technique for the detection of heparin, protamine and other biomacromolecules. However, blood electrolytes such as lipophilic anions thiocyanide (SCN−) and salicylate (Sal−) strongly interfere with the polyion response. In our present wotk, a polycation-selective polymeric membrane electrode using dinonylnaphthalene sulfonate as ion exchanger has been developed as a protamine controlled-release system for potentiometric detection of heparin. The incorporation of tetradodecylammonium tetrakis(4-chlorophenyl)borate as a lipophilic salt in the membrane dramatically improves the sensor’s selectivity towards protamine over sodium ions via influencing the activity coefficient of protamine in the membrane and a stable potential baseline can be obtained in the presence of an electrolyte background. The electrostatic binding interaction between heparin and protamine decreases the concentration of free protamine released at the sample-membrane interface and facilitates the stripping of protamine out of the membrane surface via the ion-exchange process with sodium ions, thus decreasing the membrane potential. Under optimal conditions, the proposed polymeric membrane electrode exhibits a linear relationship between the initial slope of the potential change and the heparin concentration in the range of 0.025 - 1.25 U mL-1 with an improved detection limit of 0.01 U mL-1. The presence of thiocyanide and salicylate does not interfere with the detection of heparin. This methodology makes a new pathway to use polyion-conditioned polyion-sensitive membrane electrodes for measuring target analytes. 3．Potentiometric sensing of protamine based on in situ generation and detection of sensing element. Cardiopulmonary bypass (CPB) poses great risks for hypercoagulable patients and requires management techniques to ensure an optimal dose of protamine free from thrombotic events. However, blood electrolytes such as potassium ions interfere with the detection of protamine. Based on the achievement in the development of protamine-conditioned polycation-sensitive membrane electrode for potentiometric trace-level detection of heparin, polyanion-conditioned polymeric membrane ion-selective electrode exhibiting high selectivity and excellent sensitive for determination of protamine is described in our present work. Detailed experimental results reveal that there is a linear dependence of the initial slope of the EMF change on the concentration of protamine in the range of 0.5-10 μg mL-1, with a detection limit of 0.1 μg mL-1. It is anticipated that this novel strategy will lay a foundation to develop ion-selective microelectrodes for measuring analytes in living cells.4．Potentiometric sensor for sensitive and selective detection of heparinAlthough many synthetic organic carriers with remarkable ion selectivities have been used successfully as active hosts of varius liquid membrane electrodes, carriers based on biomacromolecules have been rarely reported. In our present work, a polymeric membrane ion-selective electrode for determination of heparin is described in this paper. It is based on protamine as sensing element for selective recognition. The larger response is basically based on the electrostatic binding interaction between heparin and protamine. The electrostatic binding interaction decreases the concentration of protamine at the sample-membrane interface and further facilitates the stripping of chloride out of the membrane surface due to the charge balance. Therefore, a further increase in response toward heparin is observed. The proposed membrane electrode shows high sensitive to heparin and the response to heparin is linear in the concentration range of 0.01-0.4 U mL-1 with an improved detection limit of 0.005 U mL-1. The presence of thiocyanide and salicylate does not interfere with the detection of heparin. It is anticipated that this novel strategy will open up a broad range of possibilities in the use of protein macromolecules as sensing elements for potentiometric sensors.5．The preparation and the application of Anodic alumina nanochannels. As an important part of nanoscience, nanochannels technique has become a new growth point. Anodic alumina nanochannels (AAN) have been used for separation and detection of analytes owing to their attractive features including adjustable aperture, narrow distribution range and ease of modification.Various chemical sensors have been prepared using AAN for molecular recognition followed by appropriate signal transductions including capacitance, impedance, amperometry and spectroscopy. Potentiometric sensors are generic and highly successful approaches to chemical sensing, and AAN-based potentiometric sensors have been rarely reported. In this thesis, the highly ordered AAN with hexagonal pore structure and nanochannel diameters of 50 nm were fabricated by two-step anodization. Avidin-biotin system was chosen as a model for feasibility analysis. At last, this concept is successfully evaluated with avidin and offers potentiometric detection limit about 0.1μg mL-1. It is anticipated that this novel strategy will lay a foundation to develop nanochannel-controlled ion fluxes of polymeric membrane ion-selective electrodes for potentiometric biosensing.6．A nanochannel membrane-based potentiometric immunosensor. Development of label-free immunosensors with capability for rapid, sensitive detection of infectious diseases, continues to be an important subject for research and development. In this part, we describe a nanochannel membrane-based electrochemical immunoassay sensor for quantitative label-free human IgG analysis.. The sheet anti-human IgG was modified onto the inner wall of Anodic alumina nanochannels by glutaraldehyde interlinking method and the antibody-antigen interaction was monitored by measuring the ion fluxes through nanochannels. The sensing mechanism depends on the blocking of pore channels when the protein antigen molecules bind to antibody molecules attached to the channel walls, impeding the diffusion of indicator ions towards the interface of ISE. Macro-command-controlled procedures for switching between the potentiostatic and galvanostatic steps were designed for controlling ion fluxes through pore channels and the ISE membrane works as a transducer for sensitive potentiometric detection. Note that modification of the AAN has some problems, such as microheterogeneity. Accordingly, it still remains a great challenge to improve the repeatability, linear range and detection limit of the sensor.
|陈燕. 基于离子通量调控的电位型生物传感器研究[D]. 北京. 中国科学院研究生院,2012.|
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