【作者】 李菲菲；

【导师】 姜玮；

【作者基本信息】 山东大学 ， 分析化学， 2007， 硕士

【摘要】 本论文共包括三部分：第一部分为前言；第二部分为论文的第一章，即扫描电化学显微术“浸笔法”构建辣根过氧化物酶的微米点；第三部分为论文的第二章，即全内反射荧光显微术检测单个鼠IgG分子。在前言中对扫描电化学显微术构建图形的发展现状以及单个生物分子的检测进行了综述。第一章我们将原子力显微镜中的“蘸笔式”纳米刻饰技术（Dip-Pen nanolithography，DPN）的理念运用到SECM中，用扫描电化学显微术“蘸笔法”构建了辣根过氧化物酶（HRP）微米点。本章中我们首先研究了生物素化的HRP（Bio-HRP）在铂电极上的吸附及脱吸特性，确定了在Bio-HRP铂电极上吸附及脱吸的最佳条件。吸附条件：电极在Bio-HRP溶液中浸泡5次，每次4mir；脱吸时间：60 min。在构建HRP微米点时，先根据Bio-HRP在铂电极上吸附的最佳条件，将Bio-HRP吸附到自制的铂微米电极上，然后将铂电极在合适的电位下即-0.3 V（vs.Ag／AgCl）逼近到链霉亲和素化的基底上方10μm左右，再将Bio-HRP从电极上自然脱吸，通过生物素与亲和素的反应将其固定到基底上得到微米点，并用SECM对其活性进行了表征。同时在工作中为了减小HRP扩散所引起的微米点的扩大，在UME与基底之间形成一厚度为10μm左右的液膜，减小了分子扩散对结果的影响。第二章用全内反射荧光显微术对鼠IgG在单分子水平上进行了免疫分析。先将盖玻片硅烷化，在盖玻片表面形成环氧基团，然后在盖玻片上加上鼠IgG溶液进行孵育，使鼠IgG分子固定在玻片表面，加上BSA溶液将玻片未固定鼠IgG分子的地方封闭，然后将Alexa488标记的羊抗鼠IgG（H+L）溶液滴加在有鼠IgG溶液的地方进行孵育，这样就在有鼠IgG分子的地方接上了Alexa488标记的羊抗鼠IgG（H+L）分子，孵育完后将多余的Alexa488标记的羊抗鼠IgG（H+L）分子洗掉，用全内反射荧光显微镜检测Alexa488标记的羊抗鼠IgG（H+L）分子，通过检测到的Alexa488标记的羊抗鼠IgG（H+L）分子的个数来确定鼠IgG分子的量。本文通过去除杂质及降低所使用材料的荧光背景，使用全内反射隐失场激发，减小激发体积，并使用适宜的滤波片，采用高灵敏度的检测器ICCD对单个羊抗鼠IgG（H+L）分子进行成像，具有非常高的灵敏度，在2.0×10^-14mol／L-30×10^-14mol／L的浓度范围内，检测到的单分子的数目与溶液浓度有良好的线性关系。更多还原

【Abstract】 This thesis includes three parts. The introduction; the fabrication of micropatterns of HRP by the means of SECM "Dip-Pen" technique; the detection of rat IgG molecules by total internal reflection fluorescence microscopy.In the introduction,we introduced the evelepment of the fabrication of micropatters by SECM and single molecule detection.In chapter one, micrometer dots of HRP were fabricated by the means of SECM "Dip-Pen" technique. We utilized the elements of "Dip-Pen nanolithography" （DPN） of AFM in SECM in this work. First, the adsorption and desorption of Bio-HRP on platinum electrode were investigated by electrochemistry method. when the platinum electrode was dipped into the HRP solution for five times, and each time for 4 min, the Bio-HRP can be best absorbed; when the electrode which absorbed Bio-HRP was dipped into the PBS buffer for 60 min, the Bio-HRP can be best desorbed. Second, Bio-HRP was adsorbed on self-made platinum microelectrode in the best condition which was selected before. Then the electrode was approached to the streptomycin avidin modified substrate with -0.3 V （vs. Ag/AgCl） which was selected previously. Finally, Bio-HRP was desorbed from platinum microelectrode and reacted with streptomycin avidin, therefore the micropatterns of HRP were fabricated. The activity of the pattern was characterised with SECM. In addition, a thin film made between the microelectrode and the substrate was used to minimize the diffusion of the HRP in solution.In chapter two, the rat IgG molecules were detected by total internal reflection fluorescence microscopy. First, coverlips were dealed with glycidoxypropyltrimeth-oxysilane and epoxy group was created on the surface of coverlips; second, the rat IgG solution was added on a coverlip hatching by which the rat IgG molecules were fixed onto the coverlip; third, the coverlip was dipped into BSA solution hathing by which the remanent where having no rat IgG molecules was obturated; fourth, Alexa 488 goat anti-rat IgG（H+L） solution was added on the coverlip where the rat IgG solution was added hatching; fifth, the excrescent Alexa 488 goat anti-rat IgG（H+L） molecules was washed. After these approaches, where there is a rat IgG molecule there is a goat anti-rat IgG（H+L） one. At last, the Alexa 488 goat anti-rat IgG（H+L） molecules which were fixed onto the coverlip were detected by total internal reflection fluorescence microscopy. We can educe the amount of rat IgG molecules via the amount of goat anti-rat IgG（H+L） molecules. In this chapter we reduced all the material’s background fluorescence which was used in the experiment. using total internal reflection fluorescence microscopy, reducing excited volume, making use of fitting cubes, using ICCD to image molecules which was very sensitive, increasing detection sensitivity. The molecule number is line with rat IgG concentration in the range of 2.0×10^-14 mol/L-30×10^-14mol/L.更多还原