DNA纳米自组装结构的构建及其在信号放大生物传感与成像分析中的应用摘要DNA具有序列可设计性、简单的合成和改性、多样性以及良好的生物相容性，广泛用作纳米材料，用于构建一系列的DNA纳米结构。本文主要基于DNA自组装技术构建DNA纳米结构，与荧光、电化学，比色，光热等分析方法相结合，实现对生物活性物质，癌症标志物，pH等灵敏性，特异性检测，并应用于生物成像等领域。本论文主要开展了以下三方面工作：一、靶引发动态发夹自组装用于活细胞内mRNA及癌基因信号放大原位成像分析提出了一种基于靶分子引发的动态发夹自组装策略，用于构建DNA纳米结构，实现miRNA-21和乳腺癌易感基因BRCA1的信号放大检测以及活细胞中niRNA的原位成像分析。与传统的催化发夹组装技术相比，本工作设计了特殊结构的发夹DNA,并且发夹DNA环部设计了“spacer”结构，可以有效防止目标miRNA被取代，从而确保得到自组装DNA纳米刷结构。在未引入目标iRNA的情况下，发夹DNA可以在溶液中以亚稳态共存。当加入目标miRNA后，引发toehold介导的链置换反应，在等温条件下自组装构建DNA纳米刷。我们提出的动态组装策略具有良好的特异性，可以区分单碱基错配的目标iRNA。进一步将该方法用于活细胞中niRNA-21的荧光成像分析，实现不同细胞中miRNA-21表达水平的监测，在生物传感、生物成像、癌症等重大疾病早期诊断等领域具有广阔的应用前景。二、基于滚环扩增制备pH敏感的DNA分子聚集体构建电化学双信号生物传感器pH在人体的生命活动起着十分重要的作用，因而pH响应系统成为近年来的研究热点。在此，我们提出了一种用于pH检测的电化学双信号生物传感器。利用DNA滚环扩增技术，有效地合成了大量pH响应型长单链DNA(SsDNA),并通过酰胺键将SsDNA固定在磁珠上。长SSDNA与短链DNA(L0)杂交生成具有单双链交替的DNA纳米结构。双链部分为阿霉素(DOX)提供了大量的结合位点。当pH降低时，DNA纳米结构中单链部分具有酸性响应特征可以折叠成i-moif结构，从而导致双链部分解链，同时释放DOX。经磁分离后，可获得包含L0和DOX的溶液。与此同时，在电极表面修饰GQDs,通过酰胺反应与捕获探针结合，并通过π-π相互作用吸附DOX。当磁分离的溶液滴加到电极上，DOX即可吸附在电极表面，而L0触发催化发夹组装，将亚甲基蓝(MB)修饰的信号探针固定在电极表面上。因此，开发了一种电化学双信号生物传感器，通过检测DOX和MB的电化学信号实现pH监测。磁分离技术大大降低了检测背景，从而显著提高了检测灵敏度。三、基于滚环扩增自组装DNA花型结构封装石墨烯量子点TMB构建光热外祕泌体传感器在本工作中，我们提出了一种基于滚环扩增(RCA)自组装DNA花型结构封装模拟酶光热检测外泌体的方法。通过RCA反应制备封装石墨烯量子点模拟酶3,3',5,5'-四甲基联苯胺(GQDzyme/TMB)的微米级DNA花型结构，将外泌体适体的互补序列设计进入环状模板中，实现了外泌体适体的线性扩增。外泌体的另一条适体固定到96孔板底部，通过外泌体与适体间的特异性识别作用将外泌体捕获到96孔板内，进而与封装模拟酶的DNA花型结构结合，形成适体-外泌体-DNA花三明治结构。在HO2存在时，GQDzyme催化TMB氧化，而氧化的TMB不仅表现出颜色变化，还具有近红外激光驱动的光热效应。氧化的TMB可作为一种新型灵敏光热探针，通过近红外激光驱动的光热效应实现对外泌体的特异性和灵敏性检测。关键词：滚环扩增技术；DNA自组装；光电生物传感器；信号放大；核酸适体AbstractDNA has sequence designability,simple synthesis and modification,diversity,andgood biocompatibility,which widely used as nanomaterials to construct a series of DNAnanostructures.This article mainly build DNA nanostructures based on DNAself-assembly technology,combined with fluorescence,electrochemical,colorimetric,photothermal analytical methods to achieve sensitive and specific detection ofbiologically active substances,cancer markers,pH,even appling in the fields ofbiological imaging.This thesis carried out the following three works:1.Target-triggered dynamic hairpin assembly for signal amplification of microRNAand oncogene and its application in live-cell imagingHerein,a target-triggered dynamic hairpin assembly (T-DHA)strategy is presented,in which the hairpins can metastably coexist until the introduction of initiator(miR-21 asa model)to trigger a cascade of toehold-mediated strand displacement reaction.Unliketraditional catalytic hairpin assembly (CHA)events,a spacer structure is designed in thehairpin to prevent the displacement of the initiator during assembly,leading to anisothermal construction of DNA nanobrushes.In the absence of the target miRNA,hairpin DNA can coexist in a metastable state in solution.Once miRNA is added into thesolution,a toehold-mediated strand displacement reaction is triggered and a DNAnanobrush is constructed by self-assembly under isothermal conditions.Our proposedtarget-triggered dynamic hairpin assembly strategy has good specificity and candistinguish single-base mismatched target miRNA.The strategy further applied to thefluorescence imaging analysis of miRNA-21 in living cells,achieving the monitoring ofmiRNA-21 expression levels in different cells.Overall,our proposed target-triggereddynamic hairpin assembly strategy holds great promise not only in amplified biosensingand bioimaging,but also in clinical applications for early diagnosis of diseases,especiallycancers.2.Preparation of pH-sensitive DNA molecule aggregates based on rolling circleamplification for construction of electrochemical dual-signal biosensorpH plays a significant role in various biological pathways in human bodies.Thus,pH-responsive systems become a research hotspot in recent years.Herein,we proposedan electrochemical dual-signaling biosensor for pH monitoring.Taking advantage ofrolling circle amplification (RCA),a large number of pH-responsive long single stranded