肺癌相关长链非编码RNA电化学基因检测及其功能研究

Long noncoding RNA (lncRNA) promises to become a novel biomarker for the early diagnosis, prognosis and therapy of lung cancer. Although the present research methods on lncRNA of gene microarray technique and fluorescence in situ hybridization have the advantage of high flux, they are time-consuming and expensive imager system is also needed. In this proposal, we will adopt the means of electrochemical sensing to realize the highly effective, sensitive and rapid lncRNA detection. The sensing interface for lncRNA recognition based on three dimensional carbon nanocomposites can strengthen the localization of cDNA oligonucleotides and the hybridization efficiency of cDNA-lncRNA. The design of multi signal amplification strategy can enhance the sensitivity and decrease the detection limit. The preparation of various nano probes can realize the visualization and multiple detection of the gene sensor. Meanwhile, the constructed electrochemical gene sensor will be applied to determine the lncRNA concentrations in real samples, such as lung cancer cell or tissue extract, blood, urine and saliva, etc. Combined the data from gene sensor with the results from clinical diagnosis and lncRNA gene chip detective system, the mechanism of lncRNA in the initiation and progression of lung cancer will be demonstrated. We expect to reveal the regulation of lncRNA and provide supplementary references for realizing the gene therapy of lung cancer.

长链非编码RNA（lncRNA）有望成为肺癌早期诊断、预后判断和疗效预测的新型标志物。目前常用的lncRNA研究方法为微阵列技术和荧光原位杂交，虽有高通量的优势，但需要昂贵的成像仪器且耗时较长。本项目拟采用电化学传感手段实现lncRNA高效、灵敏、快速检测：基于三维碳纳米复合材料构建lncRNA识别的传感界面以增强cDNA链的定位性和提高cDNA-lncRNA的杂交效率；设计多重信号循环放大策略以提高检测灵敏度和降低检测限；合成多种纳米探针标记物以实现传感器的可视化和多重检测。同时，将构建的电化学lncRNA基因传感器应用于肺癌细胞或组织提取液、血液、尿液等实际样本中lncRNA含量的测试。综合临床诊断和lncRNA芯片检测的结果，论证肺癌发生发展中lncRNA对肺癌的作用新机制。研究将揭示lncRNA在肺癌中的调控作用，为实现肺癌的基因治疗提供补充依据。

本研究采用电化学传感手段实现了lncRNA高效、灵敏、快速检测，构建了一系列功能传感器。（1）建立了基于目标引发的三重等温级联放大(3TICA)策略，通过目标循环、剪切复制反应和DNA酶催化，可简单高效地检测肿瘤标志物miR-21，检测限低至0.5 aM。（2）基于二硫化钨/枝状金纳米的3D结构修饰金电极（3D WS2/DGN/AuE），结合酶辅助目标循环信号放大与DNA链杂交反应（HCR）信号增强策略，构建超灵敏电化学基因传感器用于MEG3的特征序列片段的同时检测，检测限为0.3fM。（3）采用牺牲模板法构建了三维有序大孔石墨烯/聚苯胺 (3DOM RGO/PANI)纳米复合膜，用于固定NSE-Ab，展示了很强的电子传递性能，而且PANI也被用作了电活性探针。对NSE的线性响应范围为0.5 pg/mL-10.0 ng/mL，检测限为0.1 pg/mL。（4）以3DOM MoS2–Au电极作为传感界面，SiO2@Fe3O4连接辅助DNA1（H1）/辣根过氧化物酶(HRP)作为信号探针，构建了一种电化学适配体传感器并用于黄曲霉毒素B1（AFB1）的检测，检测限低至0.01 fg/mL。（5）以miR155为检测对象，利用荧光共振能量转移（FRET）技术，以核壳CdSeTe/ZnS为信号探针，设计了目标循环信号放大策略，实现miR155高灵敏检测。miR155可引发酶切催化反应，由于AuNPs@SiO2的FRET猝灭作用，QDs的荧光信号降低，且FL强度变化值与miR155浓度成正比，检测限低至3 aM。（6）以天然桑蚕丝纤维为模板，制备了ZnO纳米管（ZnO-NTs），结合AuNPs后，得到AuNPs/ZnO-NTs复合纳米管。我们发现AuNPs/ZnO-NTs复合材料对H2O2有良好的催化活性，检测限为0.1 μM。（7）使用无模板水热法合成镍锡硫前体，进行煅烧后得到花状NiO-SnO2复合材料（NSNF），构建的无酶葡萄糖传感器检测限为1 μM。纳米材料的参与、多种信号放大手段的联用，实现了目标物的高灵敏检测；生物元件自身的高特异性目标识别也带来了实际应用意义，所有构建的电化学lncRNA基因传感器应用于血清样品中目标物检测都获得了良好的回收率结果。