外泌体介导LSR等多标志物的温致增敏型电致化学发光联合分析及其作为上皮性卵巢癌诊断方法的研究

This study is designed to integrate membrane protein LSR targeted identification units and electrochemiluminescent tracer units to obtain multifunctional probe, whose response could be enhanced by alternating temperature. The capture electrode for carcinogenic exosomes is constructed based on functional carbon nanohorns assembly. By means of the sensing mode of the sandwich type, the captured electrode is combined with the carcinogenic exosomes and multifunctional probes. Through the photothermal effect and light source modulation, the sensitized electrochemiluminescent signal is obtained which reflects the expression of the outer secretion membrane protein of LSR. To further alternating light source, the temperature of the multifunction probe continues to rise, and the disruption of membranes of exsomes is induced. Two sensing interfaces are designed near the capture electrode. Combined signal amplification sensing strategy, electrochemiluminescence signal coding technology and signal differentiability of spatial resolution electrochemiluminescence array, the quantitative detection of CA125 and HE4 in exosomes is realized. In combination with the sample analysis, the feasibility and clinical significance based on LSR, CA125 and HE4 of exsomes as the biomarkers of ovarian cancer are discussed. This will promote the study of the early diagnosis method based on exosome and provide a new method for early diagnosis and treatment of ovarian cancer.

本研究拟设计、集成外泌体膜蛋白LSR靶向识别单元和电致化学发光示踪单元，获得具有上皮性卵巢癌源性外泌体膜蛋白靶向识别能力的温致增敏型多功能探针。基于功能化碳纳米角组装体构建癌源性外泌体的捕获电极。通过“类三明治”识别模式，捕获电极依次识别并结合癌源性外泌体和纳米金多功能探针。借助光热效应，通过光源调制，获得反映外泌体膜蛋白LSR表达情况的增敏型电致化学发光信号。进一步调制光源信号，致使该多功能探针温度持续升高，诱导固载的外泌体裂解。在捕获电极附近设计两个传感界面，结合信号放大传感策略、电致化学发光信号编码技术和空间分辨电致化学发光阵列的信号可区分性，实现对外泌体胞浆内CA125和HE4的定量检测。结合样本分析，探讨上皮性卵巢癌外泌体所介导的LSR、CA125和HE4等标志物作为联合检测指标，早期诊断卵巢癌的可行性及其临床意义。这将推动外泌体介导的早期诊断方法的研究。

卵巢癌是死亡率最高的妇科恶性肿瘤。研究敏感度、特异性更高的上皮性卵巢癌的早期诊断生物学标志物便成为亟待解决的问题。本项目从组织学、体外实验和体内实验等三个角度，确认脂解激活脂蛋白受体(LSR）与上皮性卵巢癌之间的相关性，为卵巢癌早期诊断、预后判断及治疗找到了新的突破方向。项目组首次开发了基于光热调制增敏型的电致化学发光检测系统以及多种多功能探针，相关研究成果证明基于光热调制增敏型的电致化学发光检测系统具有极高的灵敏度，为获得高灵敏度的电化学发光信号提供了全新的策略。利用相关研究成果，实现了对于包括LSR等在内的多种重要目标物的高灵敏及多模式检测。项目组掌握了癌源性外泌体的提取方法。以癌源性外泌体为研究载体，通过制备一种温致增敏型多功能探针，在空间分辨电致化学发光传感平台上，实现对外泌体所介导的LSR等三种上皮性卵巢癌标志物的电致化学发光联合检测。最终结合样本分析，探讨上皮性卵巢癌外泌体所介导的LSR等标志物作为联合检测指标，识别早期卵巢癌生物学标志物的可行性。评价本项目中所建立起的系列方法对上皮性卵巢癌的敏感性、特异性等在内的诊断效能，并进一步对外泌体所介导的各标志物联合检测用于上皮性卵巢癌诊断的真实性、可靠性和实用性进行评价。项目执行期间，取得大量创新成果，项目负责人以通讯作者在Analytical Chemistry，Biosensors and Bioelectronics等国际SCI主流刊物上发表论文31篇，申请发明专利23项；先后培养研究生16名，本科生38名。相关研究将丰富电致化学发光检测的增敏模式，为上皮性卵巢癌的临床诊断和预后提供新的诊断方法，为在外泌体分析层面上了解生物医学相关的生化和分子生物学现象提供新的维度。