脱铁蛋白标记的电化学方法联合检测肺癌血清microRNAs的应用研究

The incidence and mortality of lung cancer rank first in various tumours. Effective early diagnosis is an important way to improve the lung cancer survival. It have been reported that，simultaneous detection of serum miRNAs (microRNAs, miRs) could be used as biomarkers for early lung cancer diagnosis owing to its stable expression of tumor patient. Compared with traditional methods, simultaneous electrochemical analysis of multiplex on one capture interface, which has been widely used in the rapid detection of biological and chemical molecules, displays higher efficiency and sensitivity. It could provide a new strategy for simultaneous detection of serum multi-miRNAs. Currently, electrochemical detection based on one capture interface has not been implemented in simultaneous detection of serum multi-miRNAs. In our previous study we used an electrochemical analysis technique to detect miRNA-155 rapidly and accurately. Aim to improve simultaneous detection of multi-miRNAs , the present research focused on the following miRNAs simultaneous electrochemical detection: miR-1254, miR-574-5p, miR-21, miR-155. Firstly, we establish a capture interface on one electrode surface with four kinds of nucleic acid probes to recognize miRNAs specifically and respectively. Subsequently, the detection probes were labeled by apoferritin (pH-sensitive) in which four metal irons (Cu2+, Pb2+, Zn2+, Cd2+) were wrapped respectively. Four independent stripping peaks could be observed. Finally, to achieve the ultra-sensitive detection of miRNAs, rolling circle replication and nanotechnology were utilized to enhance the response signals. The concentration of miRNAs could be calculated according to the peak current. This research can provide novel molecular targets and technical support for early lung cancer diagnosis.

高效灵敏的早期诊断是提高肺癌生存率有效途径。研究表明，肿瘤患者血清microRNAs（miRs）水平稳定，联合检测血清miRs可作为肺癌早期诊断的指标。高效超敏的基于同一敏感界面电化学分析法广泛用于生化分子等快速检测，为血清miRs联合检测提供新途径。该方法用于血清miRs联合检测未见报道。我们前期采用电化学方法实现miR-155快速分析。本项目拟以非小细胞肺癌血清miR-1254、miR-574-5p、miR-21、miR-155为研究对象，实现多组分miRs联合检测：①在一个电极表面同时固载4种核酸探针构建敏感界面，特异识别miRs；②采用对pH敏感的脱铁蛋白（包裹Cu2+、Pb2+、Zn2+、Cd2+）标记检测探针，获得相互分离的四个溶出峰；③结合滚环复制和纳米技术增强响应信号，实现四种miRs超灵敏同时检测。该方法的成功构建为非小细胞肺癌高效灵敏的早期诊断提供分子靶点和技术支持。

高效灵敏的早期诊断是提高肺癌生存率有效途径。联合检测患者血清microRNAs（miRNAs, miRs) 可作为肺癌早期诊断的指标。高效超敏的基于同一敏感界面电化学分析法广泛用于生化分子等快速检测，为血清miRs联合检测提供新途径。本项目以非小细胞肺癌血清miR-1254、miR-574-5p、miR-21、miR-155为研究对象，实现多组分miRs联合检测。目前主要完成以下工作：① 合成了系列纳米材料：金属及金属氧化我也纳米材料、石墨烯复合纳米材料。② 设计制备基于纳米技术的信号增强型电化学免疫传感器。③ 设计制备了信号增强型电化学DNA和microRNA传感器，实现对单组份核酸分子的检测。④ 采用脱铁蛋白包裹不同金属离子的策略，成功运用纳米技术、生物放大技术构建了多组分microRNA同时检测的电化学传感器，实现了同一敏感界面4种肺癌相关血清microRNA同时检测的电化学分析方法，提高了检测效率和灵敏度，降低了检测成本，为非小细胞肺癌高效灵敏的早期诊断提供技术支持。项目研究期间，发表标注了NSFC标注号的SCI收录论文1篇，中文核心期刊论文3篇，其它期刊论文3篇。另有两篇SCI论文正在投稿中。