【作者】 宋盼淑；

【导师】 童爱军；

【作者基本信息】 清华大学 ， 化学， 2013， 博士

【摘要】 荧光分子探针具有简单直观、灵敏度高、选择性好、有良好的时间和空间分辨能力等优点，已成为包括离子、小分子、生物大分子、病原体等各种与环境、生物、医学相关的分析物检测的重要手段。理想的荧光分子探针需具备高特异性的识别单元以及具有优秀荧光性能的信号响应单元，即荧光团。因此，本文结合功能核酸和聚集诱导荧光分子的优点，构建了新型荧光探针体系。首先，本文设计开发了以功能核酸作为识别单元的新型非共价标记催化分子信标荧光分析方法。该方法不仅具备催化分子信标法灵敏度高的优点，同时还具备含有缺碱基位点的非共价标记功能核酸方法操作简便，成本较低廉，对功能核酸的活性干扰较小等优点。通过8-17核酸酶和适体酶，该方法分别实现了对铅离子和有机小分子腺苷的高选择性，高灵敏度的分析检测：对水溶液中铅离子响应的线性范围为0-2.0μM，检测限为3.8nM；对水溶液中有机小分子腺苷的检测限为1.4μM。同时，该方法能够应用于检测生物样品中的铅离子和腺苷小分子。其次，本文以聚集诱导荧光分子作为响应单元，构建了两种基于聚集诱导荧光的新型荧光探针：1）比率型pH荧光探针，4-羧基-苯胺-5-氯-水杨醛席夫碱。利用其分子内羧基和羟基能够发生去质子化作用，使得溶液pH值在5.0-7.0变化时，体系在516nm和559nm处荧光强度的比值（I516/I559）随之明显变化，成功的实现了对细胞内pH值变化的原位荧光成像分析。2）铜离子荧光探针，双（2-吡啶基亚甲基）对苯二甲酸二肼。利用在水溶液中探针与铜离子形成2:1的络合物导致荧光淬灭的原理，实现了水溶液中铜离子的分析检测。并且，基于该探针的聚集诱导荧光性质制备了铜离子检测试纸，检测限可达4.2μM。上述两种探针均具有合成简单、选择性好、斯托克斯位移大等优点。第三，本文采用溶胶凝胶方法合成了包覆聚集诱导荧光染料的氧化硅纳米粒子。通过控制荧光染料的包覆量和反应条件，可以调控该荧光纳米粒子的量子产率和粒径。进一步将功能核酸对分析物高特异性、高结合力的识别性能与聚集诱导荧光分子高量子产率、大斯托克斯位移等优越的荧光性能相结合，成功地制备了NCL-适配体修饰的包覆聚集诱导荧光染料的氧化硅纳米探针。初步实验结果表明，该荧光纳米探针能够对核仁蛋白过度表达的人乳腺癌细胞系MCF-7细胞进行荧光标记，在癌细胞的识别和荧光标记领域具有潜在的应用价值。更多还原

【Abstract】 The development of fluorescent sensors for ions, small molecules, biologicalmolecules and pathogens have attracted great attention since fluorescent sesorsshowed advantages of ease in operation, high sensitivity, good selectivity, and hightime and spatial resolution. Ideal fluorescent sensors require both high specificrecognition unit and excellent fluorophores as signal response unit. Hence, the aim ofthis dissertation is to design and develop novel fluorescent sensors based onfunctional nucleic acids (FNAs) and aggregation-induced emission (AIE)fluorophores.Firstly, new label-free catalytic and molecular beacon (CAMB) sensors weredeveloped for sensitive fluorescent detection of small inorganic and organicmolecules. The sensor design had the advantages of both the label-free methods,which were low cost and could preserve the activity of the FNAs, and the CAMBmethods, which exhibited signal amplification via multiple-turnover catalyticreactions and ease for the rational design of aptazymes. By using the8-17DNAzyme,the new label-free CAMB sensor showed an approximately linear relationship withPb2+concentration in the range of0-2μM and a detection limit of3.8nM. By usingthe aptazyme for adenosine based on the10-23DNAzyme, our method could alsodetect adenosine successfully with a detection limit of1.4μM. In addition, applicationof this method in biologically relevant sample analysis was successfully achieved.Secondly, by the advantages of AIE fluorophores, two novel fluorescent sensorswere developed for monitoring pH fluctuations in live cells and building test paper forCu2+determination in this dissertation, respectively. In the former case, a ratiometricfluorescent pH sensor,4-carboxylaniline-5-chlorosalicylaldehyde Schiff base, wassynthesized via a facile reaction. The integration of hydroxyl and carboxyl groupsprovided the sensor with a significant fluorescence color change from orange to greenand an intensity ratio (I516nm/I559nm) enhancement when the pH increased from5.0to7.0in aqueous solution. Confocal fluorescence imaging of intracellular pH throughratiometric response was successfully achieved by using this sensor in live HepG2cells. In the later case, a novel fluorescent sensor bis (pyridin-2-ylmethylene)terephthalohydrazide for Cu2+determination was developed. The sensor molecule showed high sensitivity and selectivity to Cu2+by forming a1:2metal-to-ligandcomplex in aqueous solution. The fluorescence decrease at516nm was linearlyrelated to the concentration of Cu2+in the range of0.2-8.0μM. In addition, thedevelopment of test paper for Cu2+determination was successfully achieved. Both ofthese sensors showed advantages of ease in preparation, high selectivity and largeStokes shift.Thirdly, highly emissive fluorescent silica nanoparticles (FSNPs) encapsulatedby AIE fluorophores were developed by a simple sol-gel reaction in this dissertation.The emission efficiencies and particle diameters of the FSNPs were manipulated bychanging the fluorophore loadings and the reaction conditions. To combine the meritproperties of FNAs and AIE fluorophores, FSNPs were functionalized with NCL-aptamer, which facilitated the nanoparticles for potential applications in targetingnucleolin-overexpressed MCF-7cells.更多还原