具有荧光淬灭功能的介孔硅纳米颗粒用于超灵敏且能即时校正信号的内源性miRNA检测系统的研究

Morbidity and mortality of cancer have kept increasing, posing a serious threat to human health. Early diagnosis and treatment of cancer significantly improving the cure rate to over 80%, is of great importance. The endogenous expression level of miRNA, a tumor marker, is closely related to cancer; hence its ultra-sensitive and high-specificity detection allows early diagnosis of cancer. This project aims to develop mesoporous silica nanoquenchers (qMSNs)-based single or multiplex detection system for endogenous targeted miRNAs. qMSNs with broad-spectrum quenching function, serve as carriers for fluorescent dyes, and will be capped with the corresponding Antisense oligos (ASO). When the ASO specifically binds to the targeted miRNA, the ASO will shed from the surface of the nanoparticles, resulting in the release of a large number of fluorescent dyes, followed by generating an amplified detection signal, which enables ultrasensitive detection of miRNA in cells and tumor tissues. Notably, molecular beacon of GAPDH mRNA modified on qMSNs surfaces will allow detection of relative expression level of cellular targets by normalizing the detection signal according to the simultaneous control GAPDH mRNA signal, and the modified cell-penetrating polymer CPD will enhance cellular uptake. The modification of molecular beacon and CPD on qMSNs could minimize the signal variation caused by different cellular uptake in different cell types, thus improving the detection accuracy. The qMSNs-based detection system provides an ideal platform for detection of tumor biomarkers in a simpler, more accurate and more sensitive manner.

癌症发病率及死亡率逐年上升，已严重威胁人类健康。癌症的早期诊断及治疗可提高治愈率至80%以上，具有重大意义。miRNA作为肿瘤标志物，其表达水平与癌症密切相关，因而其超灵敏及高特异性检测对癌症早期诊断尤为重要。本项目拟制备具有广谱荧光淬灭功能的介孔硅纳米颗粒作为载体，包载的荧光小分子作为信号源，癌症相关miRNA的捕获探针作为识别物和孔洞覆盖物，构建单一及多种miRNA的检测系统。捕获探针与miRNA的特异性结合将引起捕获探针从颗粒表面脱落，从而导致大量荧光小分子释放并产生放大的检测信号，实现细胞及肿瘤组织内miRNA的超灵敏检测。在纳米颗粒表面修饰GAPDH mRNA的分子信标和细胞穿透聚合物CPD可分别引入内源参比以校正检测信号和提高细胞摄取量，并从这两方面降低由于细胞摄取量差异造成的检测信号差异，可提高检测的准确性。本项目将为肿瘤标志物的检测提供更为简便、准确且灵敏度高的新方法。

本项目的目标是设计及建立基于具有荧光淬灭功能的介孔氧化硅纳米颗粒的检测系统，实现对多种癌症相关RNA的超灵敏检测，并通过GAPDH mRNA的信号即时获取内源性RNA的相对水平。在本项目的资助下，已设计和选取目标RNA的捕获探针及包载的荧光小分子，并成功构建基于具有荧光淬灭功能的介孔氧化硅纳米颗粒的检测系统。该纳米检测系统在溶液中具有优异的检测能力，而且能有效检测细胞内源性目标RNA，并能根据内源性GAPDH mRNA的信号来即时获取RNA的相对水平。另外，在人宫颈癌肿瘤模型中已验证了该纳米检测系统具有良好的体内生物安全性。最后，通过对纳米系统的表面修饰，能有效提高系统的靶向性和稳定性。综上所述，本项目已构建出一个能够实现检测信号即时校正的单一或多种癌症相关RNA的纳米检测系统，为肿瘤标志物的检测提供一个简便、快速、准确且灵敏度高的新型检测方案。在本项目的支持下，已发表第一或通讯作者论文10篇，申请中国发明专利2项，完成3名硕士研究生和1名本科生的培养。