同步可视化检测双肿瘤标志物比率荧光传感平台的构建及肺癌早期诊断研究

The tumor markers detection has great significance in lung cancer early diagnosis, but its wide application is limited by the deficiency of technology. The development of visual detection method is the premise of evaluating the diagnostic value of markers and making them suitable for large-scale screening of lung cancer. Synchronous visual detection of dual tumor markers has significant advantages in lung screening. However, it is difficult to achieve synchronous visual detection for dual tumor makers due to the fact that the two fluorescence signals interfere with each other. This project creatively combined the fluorescence labeling of three primary color carbon dots (CDs) with CIE diagram to develop a ratiometric fluorescence sensing platform for synchronous visual detection of dual tumor markers. The sensing platform uses aptamers against two tumor markers functionalized onto green and red emission CDs as fluorescent probes, in which the complementary chains of aptamers modified magnetic nanoparticles were used as fluorescence quenching agents and separating agents, and blue emission CDs were adopted as the internal standard. DNase Ⅰ was also creatively used to amplify signals in this platform. The coordinates of the fluorescence color from the sensing system were verified in the CIE diagram, and the dual-markers synchronous visual sensing was established after data fitting. The sensing platform was used to detect the samples from animal and clinical scenarios. The diagnostic value of this sensing platform was studied by analyzing the relationship between various markers and lung cancer. The valued markers were selected to provide a basis for the early clinical diagnosis of lung cancer.

肿瘤标志物检测在肺癌早期诊断中具有重要意义，但检测技术的不足限制其广泛应用，发展便捷的可视化检测方法是评价标志物诊断价值使其用于肺癌大范围筛查的技术前提。双肿瘤标志物同步测定具有显著优势，但两种待测物的可视化信号相互干扰，很难实现同步测定。申请人依据前期研究基础，拟在本项目中创造性地将三基色碳点（CDs）荧光标记与CIE色度图结合，构建同步可视化检测双肿瘤标志物的比率荧光传感平台。该传感平台以两种待测物的适配体DNA链分别修饰青、红荧光CDs为荧光探针，修饰适配体互补链的磁性纳米粒为荧光淬灭剂和分离剂，定量蓝色荧光CDs为内标，并巧妙利用DNase Ⅰ酶循环放大传感信号。确认测定体系荧光颜色在CIE色度图中的色度坐标，数据拟合后建立双标志物同步可视化传感。并将传感平台用于动物实验和临床样本检测，评价可视化传感的实用性和多种肺癌标志物的诊断价值，研究肺癌早期诊断的新方法。

肿瘤作为全球致死率第一的疾病，引起人们广泛关注，发展肿瘤早期诊断新方法具有重要意义。肿瘤标志物检测是通过对患者血液、尿液或唾液中的肿瘤标志物进行检测，对患者损伤小，是一种较为有效的方便的早期诊断方法。单一肿瘤标志物的指示意义存在差异，为了降低假阳性，对多个肿瘤标志物检测。酪氨酸蛋白激酶7和miRNA-21均为明确的肿瘤标志物，可用于早期肿瘤检测。利用比率荧光检测多个肿瘤标志物可提高灵敏度和准确度。因此，本研究构建比率荧光传感平台bCDs / (Fe3O4-cDNA1-apt1-gQDs) / (Fe3O4-cDNA2-apt2-rQDs) 用于PTK7和miNRA-21的检测,为临床肿瘤诊断提供依据。所制备的比率荧光传感平台可用于PTK7和miRNA-21的检测。当体系中不存在PTK7和miRNA-21时，由于内滤效应，绿、红荧光均被淬灭，体系中仅有蓝色参比荧光（430 nm处）；当体系中存在PTK7时，apt1竞争结合PTK7，经磁性分离后，从Fe3O4 MNPs上脱离，不发生内滤效应，绿色荧光恢复（530 nm处），蓝色参比荧光不变，利用绿色和蓝色荧光强度比值 (I530/I430) 对PTK7进行定量检测，在0.5-100 ng mL-1和0.1-10 nM范围内有良好的的线性关系PTK7的线性方程为I530/I430=0.3027 lg[PTK7]+0.1291, r值为0.996，miRNA-21的线性方程为I640/I430=0.2853 lg[miRNA-21]+0.3487, r值为0.995以血清作为实际样品，通过标准加入法对PTK7和miRNA-21进行检测时，PTK7回收率达98.9-104.4%，miRNA-21回收率达87.1-108.7%，符合生物样品测定要求。实验结果证实该比率荧光传感平台可用于PTK7和miRNA-21的测定，且具有较好的灵敏度和选择性，经方法学验证和实际样品检测证实，该方法可应用于临床样本检测。