【作者】 葛佳；

【导师】 俞汝勤；

【作者基本信息】 湖南大学 ， 分析化学， 2014， 博士

【摘要】 生命体中蛋白质、核酸、酶活性以及生物小分子活动信息的研究对生物医学以及临床诊断和治疗有着非常巨大的意义。如何实时、动态、快速、准确地获取这些生命活动的信息，就成为了分析化学科研工作者面临的重大的挑战。为了克服这个难题，就需要我们发展一些准确性高、灵敏度高以及特异性高的定性或定量的方法，达到获取信息的目的。本论文瞄准上述挑战进行研究，基于滚环扩增技术和纳米材料发展了一系列分别以小分子、核酸、蛋白质和酶活性为检测对象的高灵敏性高选择性的生物传感技术，并通过对实际样品的分析，初步验证了这些技术的可行性和准确性。第2章中，单个细胞中microRNA的空间分布和表达水平对于考察microRNA及其调控网络的复杂性在生物学中的作用具有十分重要的意义。我们开发一种基于目标自引物等温滚环扩增技术高灵敏高选择性的原位检测肿瘤细胞microRNA。该方法，首先利用改良的microRNA固定方法，将microRNA的5’端磷酸基团与细胞中蛋白质的氨基通过EDC共价交联，达到固定microRNA的目的。然后预先制备的DNA环状探针与microRNA经过原位杂交，microRNA作为引物，在dNTPs和聚合酶的作用下进行滚环扩增反应，产生一条长的与环形DNA模板互补的单链DNA。细胞原位产生的扩增产物利用荧光染料标记的探针杂交识别，由于滚环扩增产物可以与成千上万的荧光染料标记探针杂交，从而达到目标microRNA高灵敏的可视化的检测。由于滚环扩增反应只能由microRNA游离的3’端触发，因此从根本上消除了mRNA以及microRNA前体的干扰。我们使用这种方法来实现人肝癌细胞SMMC-7721和人正常细胞株L02细胞中mir-222与mir-223表达水平的高灵敏的可视化检测，并进一步实现了单个细胞中不同microRNA的同时检测。我们认为目标触发的基于目标自触发滚环扩增原位检测方法是可靠的研究疾病相关的microRNA表达分析方法，在基础研究和临床诊断上具有很大的应用潜力。第3章中，DNA甲基化修饰作为一种重要的表观遗传修饰,在调节基因表达的过程中通过影响染色质结构, DNA构象、稳定性以及与蛋白质相互作用方式等途径来实现。DNA甲基化目前已成为多种肿瘤诊断的生物标志物。高灵敏高特异性的检测CpG岛DNA甲基化表型对于甲基化的研究和临床诊断都具有非常重大的意义。在本章中，我们将滚环扩增技术与DNA为模板形成荧光特性的银纳米簇相结合发展了一种可用于实际体系中DNA甲基化检测的方法。该方法首先将DNA经过亚硫酸盐的处理，再利用Ecoli DNA连接酶对错配的碱基对高特异性识别，在进行退火连接-变性解链的热循环过程中，亚硫酸盐处理后的甲基化的目标DNA与经过合适设计的与其具有完全互补碱基对的挂锁探针杂交，通过连接酶的作用下产生连接产物，该连接产物自身具有发夹结构，并有完整的限制性内切酶识别位点。银纳米簇DNA模板作为引物，在具有强链置换能力的DNA聚合酶的作用下自发启动聚合酶延伸反应以及链置换的恒温滚环放大反应，随后在限制性内切酶的作用下，释放出大量可以合成银纳米簇的DNA模板。在硝酸银、硼氢化钠的作用下，产生荧光信号，从而可实现目标DNA甲基化的检测。该方法设计巧妙新颖，有较高的灵敏度和选择性，检测限达6.4fM。第4章中，基于鸟嘌呤可触发DNA为模板合成银纳米簇荧光增强的性质发展了一种高灵敏检测肿瘤细胞中端粒酶活性的分析方法。在该方法中，我们设计了一条包含合成银纳米簇模板的端粒酶扩增引物序列，未存在端粒酶的时候，此模板合成出荧光信号很弱的银纳米簇。在目标端粒酶的作用下，引物进行扩增，扩增出富含G碱基的TTAGGG重复序列，此时合成银纳米簇不仅使得荧光信号有很大的增强，同时荧光发射波谱红移。这种方法实现了对Hela细胞中端粒酶活性的高灵敏检测，动态响应范围为500到50000个细胞，并且在该范围内峰荧光信号与HeLa细胞个数的对数呈现良好的线性关系，同时该方法具有较高选择性，线性范围宽，高灵敏度等优点。第5章中，基于核酸适配体探针可自组装在二硫化钼纳米片的表面上形成稳定的适配体-二硫化钼纳米片结构的生物传感器，发展了一种高灵敏的蛋白质和生物小分子的检测新方法。修饰了荧光素的核酸适配体探针自组装在二硫化钼纳米片的表面，依旧保持核酸适配体高的特异性和亲和力。这种复合结构可以作为低背景的平台用于ATP和凝血酶的检测。当靶目标不存在时，荧光探针与MoS2发生荧光共振能量转移，荧光被显著淬灭。当靶目标存在的情况下，靶物质与核酸适配体结合具有高的特异性和亲和力，诱导核酸适配体其形成更加刚性的分子结构，这种刚性结构的形成减弱了核酸适配体与MoS2的之间的范德华力作用，使得核酸适配体不能被MoS2有效的吸附淬灭，适配体探针与靶目标结合脱离二硫化钼纳米片的表面，荧光恢复。该方法设计简单、操作简便、灵敏度高选择性好，也可以通过灵活的使用不同的核酸适配体和DNA修饰上不同的荧光基团应用于生物医学领域多组分物质的同时检测。第6章中，多聚核苷激酶（PNK）对DNA的磷酸化修饰过程在许多的生理活动中起着非常重要的作用。本章以T4多聚核苷酸激酶(T4Polynucleotide Kinase，T4PNK)为模型，基于磷酸化的DNA被特异性外切酶降解结合WS2独特的吸附和淬灭性能发展了一种荧光方法用于T4PNK酶活性的测定。由于WS2与双链DNA的结合力较弱不能有效淬灭染色双链产物的荧光，因此得到较强的荧光信号。双链DNA作为T4PNK的底物，当T4PNK作用之后，双链DNA模板将被磷酸化，导致双链DNA立即被λ核酸外切酶降解,产生得到的单链DNA被WS2强烈吸附同时淬灭其荧光。WS2的超强荧光淬灭能力也为该方法提供了有较宽的线性范围和低的检测限，其检测下限为0.01U/mL。另外，该方法不仅可以对T4PNK的抑制剂进行定量表征，还适用于复杂生物环境中目标物的测定。本方法设计简单、操作方便、灵敏度高且选择性好，有望成为PNK酶检测的一种很好的选择并能应用于DNA损伤修复机理的研究。更多还原

【Abstract】 The essential information on small molecules, nucleic acids, protein and enzymes obtained accurately and sensitively has great significance for biological medicine study, clinical diagnosis and therapy. However, with the development of the scientific research, getting the dynamic information of these life processes sensitively and accurately in real time is still great challenge to analysts. Therefore, the development of strategies with high sensitivity, selectivity and accuracy is important for biomedical research and clinical diagnosis. In this thesis, a series of novel biosensing strategies based on rolling circle amplification and nanomaterials were developed for small molecule, nucleic acid, proteins and enzyme activity detection, respectively. These results primarily proved that the proposed technologies were feasible, reliable and accurate. The detailed content was described as follows：In Chapter2: The ability to detect spatial and temporal microRNA (miRNA) distribution at the single-cell level is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. We report for the first time the development of a target-primed RCA (TPRCA) strategy for highly sensitive and selective in situ visualization of miRNA expression patterns at the single-cell level. This strategy uses a circular DNA as the probe for in situ hybridization (ISH) with the target miRNA molecules, and the free3′terminus of miRNA then initiates an in situ RCA reaction to generate a long tandem repeated sequence with thousands of complementary segment. After hybridization with fluorescent detection probes, target miRNA molecules can be visualized with ultrahigh sensitivity. Because the RCA reaction can only be initiated by the free3′end of target miRNA, the developed strategy offers the advantage over existing ISH methods in eliminating the interference from precursor miRNA or mRNA. This strategy is demonstrated to show high sensitivity and selectivity for the detection of miR-222expression levels in human hepatoma SMMC-7721cells and hepatocyte L02cells. Moreover, the developed TPRCA-based ISH strategy is successfully applied to multiplexed detection using two-color fluorescent probes for two miRNAs that are differentially expressed in the two cell lines. The results reveal that the developed strategy may have great potential for in situ miRNA expression analysis for basic research and clinical diagnostics.In Chapter3: DNA methylation is an important epigenetic event for transcriptional regulation, being regarded as a biomarker for cancer. Sensitive and specific detection of DNA methylation in CpG sites of genomic DNA is imperative to DNA methylation discovery, study, and clinical diagnosis. Herein, we present a facile detection of DNA methylation by RCA coupled with fluorescent DNA-scaffolded AgNCs. After bisulfite treatment of methylated DNA, padlock probe was hybridized onto the target bisulfte treated and formed a circular probe by the E. coli DNA ligase if it was a perfect match between them. The oligonucleotides as scaffolds for the synthesis of AgNCs serve subsequently as a template for RCA. After HhaI cleavage reaction, the resultant reporter oligonucleotides can act as scaffolds for the synthesis of fluorescent AgNCs functioning as signal indicators in a label-free and environmental-friendly format.This RCA-based method exhibits excellent specificity and high sensitivity with a detection limit of6.4fM.In Chapter4: we presented a novel biosensing technology for the detection of telomerase activity in cancer cells based on the fact that guanine can trigger transformation of Ag NCs from a dark species to a bright red-emitting species. In this assay, the primer contains oligonucleotides as scaffolds for the synthesis of AgNCs. This primer can act as scaffolds for the synthesis of dark AgNCs without telomerase. The bright red-emitting clusters AgNCs can be obtained when the primer adds hexameric repeats (TTAGGG)n by the action of telomerase.The results indicated that the method could be used for sensitive determination of telomerase in a concentration range from500to50000HeLa cells. Given the simplicity, convenience of this approach, the proposed method may provide an alternative approach for the study of the telomerase activity.In Chapter5: A novel biosensor was designed for sensing of targets such as protein and small molecule based on the self assembled aptamer–MoS2nanosheets architecture. This DNA-MoS2nanosheet was constructed with aptamer labeled with fluorophore only at one end can self assembled onto the surface of MoS2nanosheet to form stable aptamer–MoS2nanosheet architecture, still keeping the binding affinity and specificity of the aptamer. DNA-MoS2nanosheets can act as a low background signal platform was used for the small molecule (Adenosine triphosphate) or protein (human α-thrombin) detection based on long-range resonance energy transfer. In the absence of target, the adsorption of the aptamer labeled with fluorophore on MoS2nanosheets makes the dyes approaching closely toward the proximity to MoS2nanosheets surface resulting in high efficiency quenching of fluorescence of the dyes and shows very low background. With the addition of target, binding of the aptamer probes to the target can release the aptamer away from the MoS2nanosheet, the quenched fluorescence is recovered significantly. This biosensor has the advantages in its superb specificity, being rapid, and convenient. Morover, aptamer–MoS2aptasensor design can be easily extended to develop a variety of probes for detection of a wide range of targets by simply changing the fluorophores and altering aptamer sequences.In Chapter6: DNA phosphorylation, catalyzed by polynucleotide kinase (PNK), plays significant regulatory roles in many biological events. Here, a novel fluorescent nanosensor based on phosphorylation-specific exonuclease reaction and efficient fluorescence quenching of single-stranded DNA (ssDNA) by WS2nanosheet has been developed for monitoring the activity of PNK using T4polynucleotide kinase (T4PNK) as a model target. The fluorescent dye-labeled double-stranded DNA (dsDNA) remains highly fluorescent when mixed with WS2nanosheets because of the weak adsorption of dsDNA on WS2nanosheets. While dsDNA is phosphorylated by T4PNK, it can be specifically degraded by λ exonuclease, producing ssDNA strongly adsorbed on WS2nanosheets with greatly quenched fluorescence. Because of the high quenching efficiency of WS2nanosheets, the developed platform presents excellent performance with a wide linear range, low detection limit and high signal-to-background ratio, the detection limit of T4PNK was0.01U mL-1. Additionally, inhibition effects from adenosine diphosphate, ammonium sulfate, and sodium chloride have been investigated. The method may provide a universal platform for PNK activity monitoring and inhibitor screening in drug discovery and clinic diagnostics.更多还原