【作者】 张浩；

【导师】 苏星光；

【作者基本信息】 吉林大学 ， 分析化学， 2014， 博士

【摘要】 近些年来，由生物学、化学、医学、电子学等多种领域互相交叉形成的生物传感器（Biosensor）越来愈受到人们的重视。它是由化学传感器衍生而来的，具备高选择性、快速分析、高灵敏度、低成本等优点。生物传感器可以快速灵敏地在生物环境中检测离子、小分子、蛋白、DNA并进行生物成像等。因此生物传感器在生命科学的研究领域中成为一个重要的研究方向。其中荧光生物传感器因其操作简单，成本低，检测速度快，灵敏度高，已被广泛应用于生物分析领域。随着医学发展和科学研究的不断深入，人们迫切需要一些低成本、高灵敏度、高选择性、无毒的荧光生物传感器。其中，一个非常重要的研究方向是探究在荧光生物传感器中纳米材料的使用。纳米材料是指由具有纳米量级的超微结构组成的功能材料。由于纳米材料结构上的特性，使得纳米材料具备一些特殊的性质，比如小尺寸效应和表面或界面效应。纳米材料的结合和利用会为生物传感器的发展提供一个崭新的途径。第一章，我们对各种纳米材料的合成和在生物传感器中的应用做了详细的介绍，并阐明了论文的研究意义和主要内容。第二章，我们提出一种基于单链DNA（ssDNA）与氧化石墨烯（GO）之间强相互作用和生物素与链酶亲合素（SA）特异性相互作用的新的荧光传感系统检测生物素。其中单链DNA探针的3′端修饰生物素而在5′端修饰6-羧基荧光素(FAM)。当体系中缺少SA和生物素时，生物素化的ssDNA完全被核酸外切酶I (Exo I)从3′端催化水解，从而检测到较强的荧光信号。然而，再加入SA后，由于SA的末端保护效应，Exo I则不能催化水解ssDNA，导致ssDNA吸附到GO表面，FAM的荧光被GO猝灭，产生较弱的荧光信号。若体系中加入目标生物素，由于目标生物素与生物素化的ssDNA竞争结合SA，导致体系荧光部分恢复。这个方法操作简单、灵敏度高、成本低廉。第三章，我们提出了一个灵敏的荧光测定方法，并通过荧光增强来检测葡萄糖的含量。羧基荧光素被标记在ssDNA上，当体系中存在葡萄糖时，葡萄糖氧化酶催化氧化葡萄糖生成过氧化氢。当有二价铁（Fe2+）共存时，通过芬顿反应生成的羟自由基（OH）会进攻FAM标记的长链ssDNA，从而导致不可逆转的DNA的氧化解离，生成短的FAM连接的DNA碎片。基于GO对标记的长短DNA猝灭能力的不同，根据荧光强度的变化可以检测葡萄糖的含量。该方法具有较高的灵敏度和选择性，此方法被成功用于血清中葡萄糖含量的检测。第四章，我们以3-巯基丙酸为稳定剂，通过回流加热的方法在水溶液中成功地使用一锅法制备出核壳掺杂型的Mn:ZnSe/ZnS量子点。我们优化了各种合成条件，并通过在人肝癌细胞HepG2中的细胞成像，研究了核壳掺杂型量子点Mn:ZnSe/ZnS的光稳定性，化学稳定性及其生物应用。第五章，我们利用双链DNA为模板合成荧光Cu纳米粒子，在此基础上设计了一种简单、灵敏、低成本且无标记的荧光检测方法用于核酸外切酶III活性的检测。在体系中dsDNA作为模板用于Cu纳米粒子的形成。核酸外切酶III能降解双链DNA，这样就会减少双链DNA模板的浓度，从而抑制了荧光Cu纳米粒子的形成。因此，通过该体系荧光强度的变化能进行核酸外切酶III活性的检测。与之前的报道相比，此方法不需要任何复杂的DNA序列设计或荧光染料的标记。同时，该方法对核酸外切酶III活性的检测表现出高的灵敏度和选择性。更多还原

【Abstract】 In recent years, biosensor, containing various fields of biology, chemistry,medicine, electronics et al, has attracted more and more attention. It is derived fromchemical sensors, which has the advantages of high selectivity, rapid analysis, highsensitivity, low cost etc. Biosensors can quickly and sensitively detect ions, smallmolecules, proteins and DNA in biological medium, and then be used for bioimaging.It has become an important research direction in the field of life science. Because ofits simple operation, low cost, fast detection speed, high sensitivity, the fluorescentbiosensors has been widely used in the field of biological analysis.With the development of medicine and scientific research, people urgently needsome low cost, high sensitivity, selectivity, non-toxic fluorescent biosensor. Amongthem, a very important research direction is to explore the use of nanomaterials influorescence biosensor. Nanomaterials refer to the functional materials that arecomposed of ultrastructure in nanoscale. Due to the characteristic structure ofnanomaterials, the nanomaterials have some special properties, such as small sizeeffect and surface effect. The use of nanomaterials will provide a new way for thedevelopment of biological sensors.In the chapter1, we described the attractive synthesis and application of grapheneand fluorescent nanoparticle in biosensor. Finally, we provided insights into utilizationof their properities for developments of novel biotechnology and the significance andcontents of this dissertation.In chapter2, we established a novel fluorescence sensing system for the detectionof biotin based on the interaction between DNA with graphene oxide and a terminalprotection of biotinylated single-stranded DNA fluorescence probe by streptavidin. Inthis system, streptavidin could bind to the biotinylated DNA that protected the DNAfrom hydrolysis by Exonuclease I. The streptavidin-DNA conjugate was then absorbed to the graphene oxide resulting in the fluorescence quenched. Upon theaddition of free biotin, it would compete with the labelled biotin for the binding sitesof streptavidin and then the Exonuclease I could digest the unbound DNA probe from3′to5′termini, releasing the fluorophore from the DNA. For the weak affinitybetween the fluorophore and GO, the fluorescence was recovered. The proposedfluorescence sensing system was applied for the determination of biotin in some realsamples with satisfactory reproducibility and accuracy. This work could provide acommon platform for detecting small biomolecules based on protein-small moleculeligand binding.In chapter3, we propose a novel approach for turn-on fluorescence sensingdetermination of glucose. By taking advantage of the super fluorescence quenchingefficiency of GO and specific catalysis of glucose oxidase, the sensitivity andselectivity of this GO-DNA sensing platform are highly satisfying. Hydrogenperoxide (H2O2) is produced from glucose oxidase catalysis oxidation of glucose. Dueto the present of ferrous iron (Fe2+) and H2O2, hydroxyl radical (OH) is generatedthrough the Fenton reaction and attacks the FAM-labeled long ssDNA to cause anirreversible cleavage by the oxidative effect of OH, producing the FAM-linked DNAfragment. Due to the weak interaction between GO and short FAM-linked DNAfragment, a restoration of fluorescence can be recorded with the addition of glucose.Finally, the GO-DNA sensing platform is successfully applied to detect glucose inhuman serum.In chapter4, Mn:ZnSe/ZnS core/shell doped quantum dots (d-dots) with3-mercaptopropionic acid (MPA) as the stabilizer are successfully synthesizedthrough a simple one-pot synthesis procedure in aqueous solution. We optimize thevarious synthesis conditions. The photostability and chemical stability have beenstudied and the resulting core/shell quantum dots are used as fluorescent label inhuman osteoblast-like HepG2cell imaging.In chapter5, we established a simple, sensitive, low cost and label-free strategy to detect the3′-5′exonuclease activity of exonuclease III (Exo III) using double-strandDNA (dsDNA)-templated copper nanoparticles as fluorescent probe. Upon theaddition of Exo III, the Exo III can digest dsDNA probe. With the decrease of dsDNAtemplates, the formation of fluorescent Cu NPs would be inhibit. Thus, thefluorescence intensity of dsDNA-Cu NPs would decrease. Compared to the previousreports, this strategy does not need any complex DNA sequence design, fluorescencedye label, modified DNA and sophisticated experimental techniques. This strategyproposes a new method to detect3`-5`exonuclease activities.更多还原