基于石墨烯增强核酸适配体光寻址电位传感器阵列的循环肿瘤细胞实时在线检测系统的研究

Circulating tumor cell (CTC) is a kind of malignant tumor cells circulating freely in the peripheral blood. CTC's livingly detection is very important to assess curative effect of a treatment, to analyze a medicine's effect , or to map out a personalized therapy program, etc.. While the activity of CTC can not be maitained by the traditional detection methods, the requirement to monitor CTC livingly in many modern biomedical researches can not be fulfiled. So it is of great significance to develop a realtime and online detection system of CTC. In this proposal, a realtime and online CTC's detection system is suggested based on the combination of the surface electrical field sensing mechanisim of LAPS (light addressable potentiometric sensor), the excelent electrochemical characteristics of the chemically derived graphene material and the speicific biological identification process between the aptamer and its target CTC. The merits of this proposal consist in three aspects which are: 1) compared with traditional LAPS, intensity of responding signal could be enhanced with the help of chemically derived graphene materials; 2) high specificity and affinity are possessed by aptamers which are used as CTC's probes at here; 3) By the integration of the micorfluidic technique and LAPS' array, CTC's multi-taget online and realtime detection could be realized. The fulfillment of this proposal could not only meet the current requirements for CTC online and realtime detection, but also provide a novel experimental technique and means for the research of cells in other biological system. It is believed that this proposal is worthy of being funded because of its practical and scientific value for the biomedical and cytological applicaitons.

循环肿瘤细胞(CTC)是外周血中自由循环的癌细胞,CTC活性监测在疗效评估、药效分析、个性化治疗等方面具有重要作用；然而，传统方法无法保持CTC的活性,无法满足现代生物医学研究对CTC监测的需要。因此，研究新型CTC实时在线检测体系具有很好的实际意义。 本项目将光寻址电位传感器(LAPS)的表面场效应敏感机制、石墨烯类材料优良电化学性能、核酸适配体(简称适配体)捕获靶CTC的生物识别过程相结合，提出了基于石墨烯增强-适配体-LAPS阵列的CTC实时在线检测体系。本项目的特点在于：1）与传统LAPS器件相比，石墨烯增强了响应信号的强度；2)以适配体作为CTC的探针具有较高的靶向性和亲合力；3）LAPS阵列与流体芯片相结合，可实现CTC多靶向点、实时在线检测。 本项目即可满足生物医学研究的当前需要，又可为细胞学研究提供新的实验技术方法，具有较好的实际意义和科学价值。

循环肿瘤细胞(CTC)是外周血中自由循环的癌细胞,CTC活性监测在疗效评估、药效分析、个性化治疗等方面具有重要作用；目前CTCs的检测分析仍以荧光标记方法为主，过程复杂、耗时费力、成本高，而且由于标记分子的引入，很可能会影响和改变细胞的结构和活性，往往会造成后续测试结果难以反映生物分子相互作用和特性的真实性。因此，发展高灵敏无标记检测技术，将是未来CTCs检测分析领域的发展方向。本项目提出的《基于石墨烯增强核酸适配体光寻址电位传感器阵列的循环肿瘤细胞实时在线检测系统的研究》，针对外周血中微量CTC检测的技术难题，以实现无标记、快速检测为目标，将为生物医学研究提供一种新的检测技术。.　　光寻址电位传感器 (light addressable potentiometric sensor, LAPS)是一种将半导体材料的光电效应和表面电场效应相结合，用于生物化学检测的一类新型生化传感器，其敏感单元为单晶硅-二氧化硅-氮化硅-生化敏感膜-电解质溶液。LAPS是一种表面电荷型生化敏感器件，通过对其中生化敏感膜的设计，可以构建各种高效的“生化=>电”LAPS检测平台，例如已报道的pH监测、肿瘤标志无检测等。.　　　本项目针对外周血中微量CTC检测的技术难题，构建了LAPS表面CTC捕获和响应的高效敏感界面，解决的关键技术问题包括：1）利用羧基化氧化石墨烯增强LAPS敏感单元表面的比表面积，增强LAPS对外周血中微量CTCs的响应能力；2）采用适配体探针构建适配体修饰LAPS（aptamer modified LAPS, apta-LAPS）界面，提高LAPS对外周血中微弱CTCs的捕获能力；3）构建了基于移动智能终端的LAPS实时快速检测系统，研发了3×3阵列式LAPS芯片阵列，实现了外周血中CTC的检测下限LOD=10个/mL，上述研究内容和成果已完成科技成果鉴定1项，达到国际先进水平，先后获得天津市科技进步一等奖、三等奖各一项，发表SCI检索论文4篇，EI期刊论文1篇，核心期刊论文4篇，授权发明专利3项、实用新型专利2项、软件著作权1项，申请发明专利5项，完成任务书规定的各项要求。