基于碳点石墨烯衍生物增强型光寻址电位传感器的甲基化芯片及其检测系统的研究

DNA methylation detection (abbreviated as methylation detection) is one of the important tools and contents in epigenetics research. Its great economic and social values have been exhibited in the fields of oncology, pharmacology, medical laboratory, and so on. . In order to solve the problems caused by bisulfite conversion in traditional methylation detection technologies which are information loss and inability of live cell methylation monitoring, the research on carbon dots-graphene derivatives enhanced light addressable potentiometric sensors (LAPS) based methylation chips and detection system is proposed in this appliction by a triple-enhancement-mechanism which is formed by the integration of the functions of LAPS to amplify surface charges, carbon dots to accelerate charge transfer and graphene derivatives to increase bio-functionalization. . A new direct electrical methylation detection method would be created in this application. Taking three kinds of circulating tumor DNA (ctDNA) as examples, the rapid and simultaneous methylation detection of multi-ctDNA could be realized. It would provide an instrumental platform to promote clinical applications of ctDNA methylation research.. The live cell methylation dynamic monitoring system would be created based on the proposed carbon dots-graphene derivatives enhanced LAPS. Furthermore, the LAPS methylation sensing mechanism, as well as the methylation identification model, in live cells would be established according to the cell impedance variation induced by methylation. And the methylation monitoring in live cells could be achieved by taking MDAMB231 cell as an example. This work would provide a novel research tool for epigenentics.

DNA甲基化检测(简称甲基化检测)是表观遗传学研究的重要工具和内容，在肿瘤医学、药物学、检验医学等多个领域都具有极大经济和社会价值。为解决传统甲基化检测中亚硫酸氢盐(BS)转化引起的信息丢失和无法实现活细胞甲基化监测的问题，本项目将光寻址电位传感器(LAPS)表面电荷放大、碳点促电荷传输、及石墨烯衍生物增强生物功能化三重增强作用相融合，研制基于碳点石墨烯衍生物增强型LAPS的甲基化芯片及其检测系统；创建免BS转化的甲基化直接电学检测新方法，以三种循环肿瘤DNA(ctDNA)为例实现ctDNA甲基化快速、同时检测,为促进ctDNA甲基化研究成果的临床应用提供技术平台；创建基于碳点石墨烯衍生物增强型LAPS的细胞甲基化动态监测系统，建立基于甲基化诱导细胞阻抗变异的LAPS-细胞甲基化敏感机制和甲基化辨识模型，以MDAMB231细胞为例实现细胞内甲基化动态监测,为表观遗传学研究提供新的研究工具。

DNA甲基化是表观遗传学的重要组成部分，其研究成果在慢性病、肿瘤医学、医学检验、药物研发、器官移植等多个领域显示出极大的经济和社会价值，然而，传统DNA甲基化检测方法操作复杂和高成本的问题，限制了甲基化检验的推广应用。针对这一问题，本项目分别利用光寻址电位传感器（LAPS）和石墨烯场效应晶体管（GFET）开展了新型DNA甲基化传感器及检测系统的研究工作。针对甲基化检测对量化被测DNA链上甲基化位点的关键问题，在传感器敏感界面的设计方面，摆脱了传统的将探针固定于芯片表面的方案，创新性提出了被测DNA链固定于传感器表面甲基化检测方案，利用DNA序列上甲基化位点与甲基化抗体之间的特异性免疫性识别作用，实现对LAPS和GFET敏感单元表面电荷调控，从而成功的实现将被测序列的甲基化信息转换为输出光电流和源漏电流的变化。利用这一策略，我们利用卟啉在芯片表面两种可能的形态，分析了DNA序列形态对甲基化检测效果的影响，以测试连为例的实验结果显示，该方案可实现对单甲基化位点的辨识，进一步与肿瘤医院结合，将这一策略用于病灶组织和健康组织DNA序列的测试，取得了较好的实验结果，相关研究内容发表在ACS Omega、Analyst等学术期刊。在检测系统研发方面，与天津市兰标电子科技发展有限公司合作开发多通道LAPS检测系统一套，成果发表于学术期刊传感器技术与应用、科技通报。进一步，在本项目的资助下开展了全碳基LAPS和神经形态传感器的探索性研究，并将其用于DNA甲基化和神经递质的检测，研究结果发表于ACS Sensors和Advance Function Material。综上所述，本项目在执行期间共发表研究型期刊论文21篇，会议论文3篇，申请发明专利1项，授权发明专利2项，培养博士研究生2人，硕士研究生7人，如期完成任务合同书的基本要求。