基于超敏电致化学发光传感器的肝癌长链非编码RNA的检测及应用研究

Studies have shown that high expression factor (HULC) and some molecules of long chain of non-coding RNA (lncRNA) spectrum of liver cancer are new diagnostic biomarkers for liver cancer diagnosis. However, related detections do not be carried out in any clinical laboratory. The key reason may be some shortages of existing methods for lncRNA detection, for instance, high equipment requirements, limitation of detection range, complicated operation or harmful. It leads to difficultly reaching out the desired requirements of clinical testing. Therefore, a novel technology for lncRNA detection with sensitive, specific, rapid and non-toxic is urgent. . According to literatures, electrogenerated chemilumineseenee (ECL) sensor has distinct advantages, for example, high sensitivity, specificity, simple and fast, and so on. SO it is a powerful pathway to solve the problem mentioned above. This project would try to study how to detect lncRNA including HULC, MALAT-1 and HOTAIR in liver cancer serum. To improve immobilization efficiency of capture probe 1, the new nano- materials of single-walled carbon nanohorns (SWCNHs) / reduction of graphene (rGO) / gold nanoparticles were used to modify the sensors. Then, to achieve a large number of lncRNA conversion fragments, fragment conversion and thermostat amplification (FCTAMP)technology will be developed. Moreover, a novel Ru (phen) 32+-DBAE ECL method will be established to amplify detection signal. High throughput detection method of lncRNA would be achieved combined with sensor array technology. Through those study, a sensitive and specific, thermostatic simple, fast and efficient non-toxic and non-invasive new technology will be established for lncRNA detection, providing innovative platform for the early diagnosis of liver cancer.

长链非编码RNA（lncRNA）谱中的肝癌高表达因子（HULC）等分子是肝癌新型诊断标志，但相关项目在临床检验中并未开展。关键原因是目前lncRNA检测方法存在设备要求高、检测范围局限或对人体有害等缺陷，难以达到临床检验的理想要求；亟需开发敏感特异、快速无毒lncRNA检测新技术。电致化学发光传感器具有灵敏度高、特异性好、简便快速等独特优势，是问题解决的有力途径。本课题拟以肝癌血清为研究对象，以HULC等为靶点；采用单壁碳纳米角/还原石墨烯/纳米金等新型纳米材料修饰传感器提高探针固载效率，建立恒温、快速、高效的核酸转换扩增技术实现lncRNA的大量扩增，构建新型Ru（phen）32+-DBAE电致化学发光技术实现检测信号放大；通过SPE电极阵列实现lncRNA高通量检测；从而建立起lncRNA敏感特异、等温简便、高效快速和无创无毒的新型检测新技术，为肝癌的早期诊断提供创新性的技术平台。

肝癌是我国的第四大恶性肿瘤，肝细胞肝癌（HCC）是主要病理类型。AFP是其诊断标志物，但由于灵敏度和特异性的缺陷导致了3-59%的漏诊率。文献报道显示，HULC等长链非编码RNA（lncRNA）在肝癌组织或血清里表达显著升高，可作为HCC的潜在标志物，是AFP的有力补充。但目前lncRNA检测方法存在检测范围局限或对人体有害等缺陷，难以达到临床检验的理想要求；亟需开发敏感特异、快速无毒的检测新技术。. 本课题针对肝癌血清，以lncRNA标志分子为靶点；研究SWCNHs-Au膜电沉积技术；制备并应用新型复合纳米材料以提高探针固载率；研究片段转换恒温扩增（FCTAPM）技术；构建新型Ru（phen）32+-DBAE等电致化学发光技术；研究基于复合纳米材料催化的多种电化学和ECL传感器以检测HULC、MALAT1等lncRNA；探索SPE阵列技术以实现高通量检测；通过这些研究建立起lncRNA敏感特异、恒温简便、高效快速的新型检测新体系。. 研究结果表明最适本项目的SWCNHs水化剂为PDDA，浓度为0.5%，超声2h。SWCNHs-Au膜电沉积的最佳条件是在含0.25% SWCNHs和0.05% HAuCl4的碳酸盐缓冲溶液（pH9.0）中沉积。此外，项目研制了空心纳米铂、石墨烯-纳米铂钯微型树枝复合物、空心纳米金铑复合材料、原位SWCNHs-空心金等纳米材料；都具有比表面积大或者催化活性好的优点。研究并建立了FCTAPM核酸扩增技术。建立了Ru(phen)32+-DBAE和gC3N4等ECL发光体系。利用丝网印刷电极技术构建模块化SPE阵列的技术；设计出微型集成传感器模型，并进行了前期验证性3D预打印。建立了多种基于贵金属纳米复合物催化并结合其他信号放大策略的lncRNA 电化学传感器或ECL生物传感器技术，分别检测了HULC、NEAT1、MALAT-1和HOTAIR等lncRNA分子，优化并定型了各型参数；在肝癌血清中验证性检测中表现了高灵敏、高特异和良好稳定性等优良性能。本项目建立的lncRNA检测新体系，有望为肝癌诊断提供新的可选技术平台；也为其他相关研究奠定了良好的技术和理论基础。. 迄今为止，本课题共发表SCI 论著2篇，申请国家发明专利7项（其中已获授权4项）；获得国家进步一等奖1项；培养硕士研究生1名。