基于功能化石墨烯的循环肿瘤细胞分选与活细胞原位荧光成像研究

This project is aiming to develop integrated systems for efficient circulating tumor cells (CTCs) capture with high-purity, sensitive CTCs detection and comprehensive molecular profiling based on nanomaterials of graphene and nanoparticles. The specific research works are listed as following: (1) Marker-free negative capture system of CTCs with high-purity will be constructed based on graphene oxide-coated magnetic nanoparticles (GO/MNPs). GO/MNPs can improve magnetic response and reduce loss rate of MNPs. Moreover, MNPs have relatively high surface to volume, benefiting to modify more antibodies, which will bring higher capture efficiency to background cells and improve the purity of CTCs. (2) Multiple-marker positive capture system of CTCs will be constructed based on multiple aptamers network functionalized graphene. The size difference between aptamer network and CTCs is small and tandem aptamers have cooperative affinity, which will bring higher binding capacity and faster binding kinetics, and thus improve the capture efficiency and reduce the absorption of background cells. (3) Non-destructive CTCs detection assay will be constructed based on CTCs genetic markers responsive “off-on” spherical nucleic acids probes. (4) The analysis of genetics and phenotypes in live cells at the single-cell level will be obtained based on multicolor spherical nucleic acids probes that designed according to genetic markers of different phenotypes CTCs. (5) The personalized treatment plans of tumor based on CTCs will be broaden through ex vivo culture and individualized testing of drug susceptibility of CTCs from different cancer patients. The development of these works will benefit to comprehensively access to tumor information and the clinical management of tumor.

本项目拟以石墨烯和纳米粒子为核心材料，高效率、高纯度循环肿瘤细胞(CTCs)捕获及整合检测和生物学特性分析为目标，开展以下工作：（1）将磁纳米粒子装载到氧化石墨烯表面，提高磁响应速度、降低丢失率，协同纳米粒子比表面积大、抗体修饰量大，构建阴性捕获体系，增加背景细胞捕获效率，提高CTCs捕获纯度；（2）利用多重适体网功能化石墨烯构建阳性捕获体系，适体网与肿瘤细胞尺寸差异小、串联适体具有协同亲和力，利于快速充分结合，增加CTCs捕获效率，减少背景细胞吸附；（3）构建CTCs基因标志物响应的“关-开”型球形核酸探针（SNA），通过活的CTCs内荧光成像实现CTCs定量检测；（4）基于不同表型CTCs标志基因构建多色SNA，根据多重特异性荧光响应，实现单个CTCs水平基因原位分析及表型分析；（5）捕获不同肿瘤患者血液中CTCs，进行培养，并结合药敏和基因分析，拓宽基于CTCs个体化治疗肿瘤研究范围

实时、原位、全面的获取肿瘤信息对于癌症的早期诊断、精准治疗、预后判断具有重要的指导价值。本项目基于DNA纳米技术和无机纳米材料设计并构建了一系列的纳米探针用于肿瘤细胞内标志物的成像及相关功能生物分子的检测等研究。基于可编程的DNA-AuNPs球型核酸探针的设计，开发了细胞内mRNA响应的DNA计算平台，实现了细胞内EMT相关mRNA和癌症相关mRNA的原位成像分析及不同表型癌细胞的识别和区分；构建了二氧化锰纳米片介导的比率荧光生物传感器用于miRNA的检测和成像，与单染料探针相比，比率荧光生物传感器可以实现两个波长下荧光强度的同时检测，避免由实验环境影响产生的假阳性信号，利用该生物传感器可以有效区分不同细胞内miRNA-21的表达水平；利用氧化锌纳米颗粒遇酸降解的特性，构建了pH响应型氧化锌纳米探针介导的DNAzyme信号扩增策略用于多重miRNAs的灵敏检测和活细胞成像，对三种细胞内miRNA-21的表达水平进行了区分，在同一种细胞内对miR-21和miR-373进行了同时成像；此外，构建了多种新型的3-D DNA walker，可用于相应目标物的灵敏、特异检测。本研究的结果，有望为肿瘤的早期诊断和精准治疗提供新的策略和技术支持。本项目共发表SCI研究论文31篇，综述1篇，其中，Chemical Science 1篇，Analytical Chemistry 2篇，ACS Applied Materials & Interfaces 3篇，Biosensors and Bioelectronics 1篇，Chemical Communications 3篇，Sensors and Actuators B: Chemical 7篇。