纳米技术的发展为肿瘤特异性生物标志物的分析与检测提供了新的发展机遇，发展基于纳米生物界面的分析技术可以帮助我们更好地研究癌症发生发展的分子机理，开发高灵敏性的癌症早期诊断方法。本文以肿瘤发生发展相关的天然纳米粒子-外泌体的标记为切入点，探究了肿瘤外泌体在体内不同器官的摄取行为和分子机制；同时将磁性纳米粒子与点击化学反应结合，构建了肿瘤相关microRNA (miRNA)分子靶基因分离和鉴定方法；发展了基于金纳米粒子的高灵敏性肿瘤标志物检测方法，主要研究内容和结论如下： 首先，我们合成了带有叠氮基团的胆碱衍生物，通过含胆碱的磷脂分子生物代谢途径将叠氮基团修饰到外泌体的膜上，并通过点击化学反应将荧光分子共价连接到外泌体膜上。通过这种标记方法，我们对三种乳腺癌细胞系(MCF-7， MDA-MB-231，HS578T)分泌的外泌体标记了不同的荧光分子并进行小鼠活体、组织成像以及组织切片免疫荧光检测分析。发现来源于MDA-MB-231和HS578T细胞的外泌体在小鼠脑部有明显富集并且可以被小鼠脑部星形胶质细胞所摄取。通过蛋白质组学与生物信息学分析，我们发现ephrinA型受体2(EPHA2)可能与外泌体脑靶向性相关，随后通过CRISPR/Cas9基因编辑系统，我们构建了EPHA2基因敲除细胞系，并利用上述标记方法对野生型及EPHA2缺失的突变型外泌体进行体内示踪以及体外摄取分析，发现来源于EPHA2缺失外泌体在小鼠脑部富集明显低于野生型外泌体，且EPHA2的缺失降低了星形胶质细胞对外泌体的摄取。 其次，我们构建了基于磁性纳米粒子的肿瘤相关miRNA靶基因富集与鉴定方法，利用兼具疏水性与可点击反应性的二苯并环辛炔(DBCO)标记miRNA分子的3’末端，通过免疫荧光共定位以及体外结合实验证实DBCO标记的miRNA分子可成功整合到RNA诱导的沉默复合体中，同时利用计算模拟结合方法，探究了DBCO标记的miRNA与Ago2蛋白的结合方式。随后将DBCO标记的miRNA作为“诱饵”以结合细胞内的靶基因，并通过DBCO与叠氮化磁性纳米粒子的点击反应将靶基因分离出来，通过己知靶基因检验此方法可行性，随后利用生物信息学分析与双荧光素酶报告检测方法，我们分离了miRNA一系列新的靶基因，并证实miR-21靶向肌型磷酸果糖激酶基因(PFKM)，降低细胞葡萄糖摄取以及ATP的产生，从而调节HEK293T细胞中糖酵解途径。 最后，我们提出了一种基于金纳米粒子(AuNPs)的高灵敏性生物标志物检测方法。将覆有AuNPs的粗糙基底与带有辣根过氧化物酶(HRP)偶联检测抗体的AuNPs结合，在增大基底上捕获抗体数量的同时，还增加了局部HRP的量，通过上述两个信号放大步骤来进一步降低检测限。基于这种连续信号放大系统，我们实现了临床样本中前列腺特异性抗原(PSA)的高灵敏度检测。 关键词：肿瘤；外泌体；microRNA；分子标记物；诊断

 The evolution of nanotechnology provides new opportunities for the analysis and detection of tumor-specific biomarkers. The development of nano/bio interface-based analytical method contributes to the deciphering of the intrinsic mechanisms under cancer development,and offers new way for the early diagnosis of cancer. Herein,we explored the molecular mechanism of organ-specific uptake of tumor derived exosomes; we then identified the intracellular target genes of tumor related miRNA by using clickable miRNA and magnetic nanoparticles; we finally developed a highly sensitive and compatible gold nanoparticle-based method for biomarker detection. The main research contents and conclusions are as follows: First, we reported that the phospholipid-based bioorthogonal chemistry was a simple and biocompatible strategy to label exosomes derived from a set of breast cancer cells. This labeling strategy was based on a two-step procedure: i) metabolic incorporation of azides into exosome phospholipids and ii) bioorthogonal conjugation of fluorescent probes with the anchored azides. After simultaneous intravenous injection of multiple fluorescently-labeled exosomes into a living mouse,their dynamics and organ-specific biodistribution were directly monitored in vivo. Impressively, we found that the exosomes derived from MDA-MB-231 and HS578T cells could preferentially target to the brain astrocytes of nude mice. With the help of mass spectrometry and bioinformatic analysis,we found that ephrin type-A receptor 2 (EPHA2) could be a key factor promoting the brain-tropic metastasis of the exosomes. We then knocked out the EPHA2 in MDA-MB-231 cells and employed the labeling strategy to explore the in vivo distribution of exosomes derived from wild-type and mutant MDA-MB-231 cells. As a contrast, the exosome derived from the EPHA2-deficient cells decreased the uptake efficiency of brain tissues. Second, we developed a biochemical tool that coupled magnetic nanoparticles with clickable moieties,to directly capture and pull down microRNA (miRNA) targets inside cells for identification. Through computational study of the molecular structure of RNA-induced silencing complexes (RISCs),we showed that dibenzocyclooctyne (DBCO) could serve as a structurally adaptive,high-affinity "bait" to recognize the Ago2 protein directly and thus to boost the formation of RISCs with miRNA target genes in live cells. In addition, the DBCO tag facilitated rapid enrichment of target genes via clickable purification. Using this approach, a series of miR-21 target genes were pulled down from HEK 293T cells and identified with qRT-PCR. This clickable "bait" strategy enabled the discovery of several new target genes of miR-21, making it a promising tool for RNA research. Finally,we presented a highly sensitive and compatible gold nanoparticles (AuNPs)-based,two-step signal amplification system for biomarker detection. AuNPs were coated onto the surfaces of 96-well plates to generate rough surfaces, which enable immobilization of many more capture antibodies than a smooth substrate. As a result,detection sensitivity was enhanced significantly. Besides, the horseradish peroxidase (HRP)-conjugated detection antibodies were labeled on large-size AuNPs, which increase the localized amounts of HRP and thus further lower the detection limit. Based on the consecutive signal amplification system, a high-sensitivity assay was achieved. This assay was allowed to detect the PSA levels in clinical samples without changing the current standard immunoassay setups,showing great potential in many settings where immunoassays are needed. Key Words: tumor; exosome; microRNA; biomarker; diagnosis