PdNi-C3N4量子点和信号级联放大的微纳传感器多联检测肝癌lncRNA标志物的研究

Some lncRNAs including HULC and MALAT1 can be used as diagnostic biomarkers for liver cancer. Multi-lncRNAs combined with AFP is an effective means for early diagnosis of liver cancer. Conventional detection methods exist detection linear limitations, toxic reagents, specificity defect, and so on. Even though the electrochemiluminescence sensor is an ideal method to detect lncRNAs for sensitive and fast advantages and also have been employed to detect HULC, MALAT1, HOTAIR by us, it is difficult to achieve multi-lncRNAs clinical application because of the toxicity of reagents, single detection channel, signal disruption. So that technological breakthrough is urgent. . In order to break the current bottleneck, this project would try to study how to detect multi-lncRNAs biomarkers in liver cancer serum. And to achieve non-toxic operation, PdNi-C3N4 quantum dot luminescence system would be developed. Then, telomerization telomeric rapid extension technology (TSHRET), which G3/hemin may catalysis C3N4 combing Pd-Ni nanocage with signal cascade synergistic effect, would be established to improve the sensitivity. Moreover, rapid preparation of WS2/Au nano-film can increase the amount of probes loading. And nano-magnetic stirring would make the reaction uniform and fast. Micro-nano ECL sensor arrays would be printed through 3D micro-nano printing technology, which can improve the specificity and achieve high throughput detection. Such a new high through analysis system with non-toxic and non-invasive, fast and efficient, sensitive and specific will be established for the detection of lncRNAs biomarkers. Through those studies, the purpose of multi-lncRNAs combining with AFP synchronization detection would be achieved. And this strategy can provide early diagnosis technology for liver cancer and also may be use as alternative options for other tumors diagnosis.

HULC和MALAT1等lncRNA是肝癌新型标志物，多联lncRNA联合AFP是肝癌早诊的有效手段。常规检测方法存在检测线性局限、试剂有毒、特异性差等缺陷。虽然电致化学发光传感器灵敏快捷，是检测lncRNA的理想手段；我们前期运用该技术也检测了HULC等，但由于试剂低毒、通道单一、信号扰乱，难以实现lncRNA多联临床应用；亟需寻找技术突破。为打破当前瓶颈，本课题拟以血清lncRNA为靶点；构建PdNi-C3N4量子点无毒发光技术，实现绿色操作；建立酶控端粒模拟快速延长技术，协同Pd-Ni纳米笼实现信号级联放大以提高灵敏度；快速制备WS2/Au纳米膜增加探针固载量；纳米搅拌则使反应匀相快速；通过3D微纳打印构建传感器阵列，提高特异度并实现高通量。从而建立无毒快捷、高敏特异的高通量检测体系，达到多联lncRNA联合AFP同步检测的目的；为肝癌提供早诊技术，也为其他肿瘤诊断提供可选方案。

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。HULC等长链非编码RNA（lncRNA）是HCC的新型诊断标志物。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。. 本课题以肝癌血清lncRNA标志分子为靶点，制备新型纳米催化材料和发光纳米物质，构建新型电化学生物传感器检测MALAT1；建立RAP扩增技术，恒温快速检测肝癌lncRNA标志物HULC和H19；研制了十万目负压自驱微流芯片并构建了数字RPA绝对定量技术体系以检测lncRNA H19；构建NHMC-dPCR检测技术以绝对定量肿瘤EGFR突变。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。. 项目组首先制备了WS2、g-C3N4绿色量子点和PNG-PAMAM等纳米材料，具有比表面积大或者催化活性好的优点。构建了SWCNHs/Au@ PNG-PAMAM多重催化MALAT1电化学传感器，具有灵敏度高、特异性和重复性好的优点；在肝癌血清中进行了有效验证。lncRNA 重组酶聚合酶（RAP）扩增技术检测肝癌血清lncRNA标志物HULC和H19表现了恒温快速、灵敏特异的特性，筛选了1~2对特异性好、灵敏度高的引物对和探针。研制了十万目负压自驱微流芯片（ NHMC芯片 ），采用构建的NHMC微流芯片对lncRNA H19进行了数字化绝对定量，H19的实际拷贝数与理论拷贝数吻合率达到96.3%，达到了理论要求；定量范围为103~108个拷贝。构建了NHMC-dPCR检测技术，并对EGFR突变G719S进行了灵敏特异的数字化分析。此外，还资助研究构建了时间分辨测流免疫层析，检测了血清中的IL-6；另外资助研究了血清部分疾病标志物与临床指标关联分析。本项目针对肝癌血清标志物lncRNA选定的靶标，开展了系列新检测技术和新检测体系研究，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5篇，中文论著5篇；申请国家发明专利8件（其中已获授权5项）；培养硕士研究生2名（已顺利毕业1名）。