基于发光金属有机骨架与量子点共传感的一体化液体活检新方法研究

Early diagnosis and dynamic evaluation of glioblastoma are of great significance for improving the survival rate of patients, but it is still difficult by using clinical techniques. Liquid biopsy, a minimally invasive and convenient method, overcomes the shortcomings of traditional methods and provides dynamic tumor monitoring. CD63 protein is highly expressed on the membrane of exosomes from glioblastoma and is a specific biomarker of glioblastoma. However, the content of exosomes is very low in blood, and it is necessary to extract exosomes before the quantification of biomakers. The extraction and detection cannot be integrated by using traditional method. In this study, a novel liquid biopsy method for simultaneous capture and determination of exosomes is proposed. The magnetic fluorescent metal-organic framework is used as adsorbent, fluorescence quencher and also fluorescence internal standard and quantum dots modified CD63 aptamer is applied as fluorescent probe. Exosomes are specifically captured by electrostatic and immune sorption of metal-organic framework. Meanwhile, the amplified fluorescent signal is converted into the concentration of CD63 protein by the formation and disappearance of photoinduced electrontransfer process of fluorescent nanosensors with highly sensitivity. The proposed method can simultaneously accomplish the capture and quantification of exosomes with the advantages of simple operations, low cost and dynamic evaluation which provides a new strategy for clinical diagnosis of tumor.

胶质母细胞瘤的早期诊断和动态评估对于提高患者生存率具有重要意义，但现有技术仍难以实现。液体活检克服传统方法的不足，提供一种微创、便捷的肿瘤动态监测手段。CD63蛋白在胶质母细胞瘤来源外泌体表面高表达，是一种特异性胶质母细胞瘤生物标志物。然而，外泌体含量甚微，需提取后进行标志物检测，无法集提取和检测于一体。本课题首次提出利用磁性荧光金属有机骨架作为提取剂、荧光猝灭剂和荧光内标，量子点修饰的核酸适体作为荧光探针，构建外泌体提取及生物标志物检测一体化的液体活检新方法。利用静电和免疫吸附作用特异性提取外泌体，同时通过荧光纳米传感器之间光诱导电子转移体系的形成和消失，将放大的荧光信号转化为外泌体CD63蛋白含量，实现高灵敏定量检测。该方法将外泌体提取及CD63蛋白定量分析一体化，具有操作简单、成本低、可动态监测等诸多优势，为临床提供一种肿瘤诊断和评估新策略。

癌症的早期诊断对于患者生存率的提高具有重要意义，研究表明特异性癌细胞来源的外泌体与肿瘤的生长和转移密切相关，可用于癌症早期诊断。但癌细胞来源的外泌体含量极低，特异性的提取和高灵敏的定量检测困难，且无法集提取和检测于一体，临床应用受到了较大限制。EpCAM是一种跨膜糖蛋白，在上皮癌变过程中发挥重要作用，在上皮细胞癌来源的外泌体上表达显著升高，可作为特异性生物标志物。本研究基于锆基金属有机骨架（Zr-MOFs）建立了乳腺癌外泌体提取和荧光定量检测平台。首先通过磁性修饰的UiO-66非特异性锚定外泌体，其后捕获的外泌体特异性识别EpCAM抗体修饰的荧光探针UiO-66-NH2，形成具有荧光信号的“MOF-外泌体-MOF”三明治夹心结构。夹心结构的荧光强度与乳腺癌外泌体含量呈正相关，该方法实现了乳腺癌细胞来源外泌体的高特异性、高灵敏的检测，且可有效区分健康志愿者和乳腺癌患者，为乳腺癌早期诊断提供新思路。此方法适用性强，可扩展至其他类型上皮癌的早期诊断。