基于超微电极的无标记均相电化学DNA传感器  

Homogeneous Label-Free Electrochemical DNA Sensor Based on Ultramicroelectrodes

作　　者：党珮珠 田璐瑶 束玉茹 郑嘉玥 陈芳明 张德文 王健[1] Dang Peizhu;Tian Luyao;Shu Yuru;Chen Fangming;Zhang Dewen;Wang Jian(Xi’an Jiaotong University,Xi'an,710049)

机构地区：[1]西安交通大学,西安710049

出　　处：《生命科学仪器》2020年第6期32-38,共7页Life Science Instruments

基　　金：西安交通大学基本科研业务费xzy012019071;大学生创新训练项目SJ201910698404;国家自然科学基金资助项目31800827。

摘　　要：特定序列核酸的检测在临床病原体检测、癌症早期诊断以及遗传病确诊等方面至关重要。无标记均相电化学传感器在低消耗、低成本、易操作、快速检测和高重复性等方面具有显著的优势。我们基于复合功能核酸分子和超微电极检测装置,构建了无标记均相电化学核酸传感器,用于PIK3CA循环肿瘤DNA(ctDNA)的高特异性检测。将PIK3CA ctDNA互补序列和G-四聚体脱氧核酶偶联,分别作为目标识别元件和信号转换元件,设计了具有茎环结构的单链DNA,其中G-四聚体和ctDNA互补序列通过碱基互补被封闭。当目标DNA即PIK3CA与其互补序列特异性结合后会导致DNA构象变化,释放G-四聚体并发挥过氧化物酶的活性,催化H2O2和对苯二酚(HQ)反应,产生对苯醌(BQ)和水。使用碳纤维超微电极微型电化学装置对微升体积样本中HQ和BQ的氧化还原电流信号进行检测。结果表明:氧化还原电流信号随目标DNA分子浓度变化而变化,检测限达20 nM,可有效区分完全互补序列和单碱基错配序列DNA。此外,在血清掺杂样本中该传感器表现出类似的检测效果。本研究设计的电化学生物传感器实现了微升体积内ctDNA的高性能检测,为特定病原体或肿瘤基因的靶向DNA序列检测提供了一种新思路,在医学诊断和便携式检测中具有广阔的应用前景。The detection of specific sequence of nucleic acids is of significant importance in clinical pathogen detection,early diagnosis of cancer and diagnosis of genetic diseases.Among the various detection techniques,homogenous label-free electrochemical assay showed excellent performance like low cost,easy operation,rapid detection,and high repeatability.In this work,we designed a homogenous label-free DNA sensor based on the combination of ultramicroelectrodes and functional nucleic acid for the sensing of PIK3CA circulating tumor DNA(ctDNA).The functional nucleic acid consists of a G-quadruplex sequence and a complementary sequence of PIK3CA ctDNA,which were both elaborately blocked via base pairing in a stem-ring structure.The presence of ctDNA can trigger the conformation switching and therefore released G-quadruplex part and turned on DNAzyme activity.The carbon fiber electrode based miniaturized electrochemical device was applied to the detection of homogeneous DNAzyme catalytic activity at the microliter level.The proposed DNA sensor showed dose-dependent responses to PIK3CA with high specificity,which provided a simple and rapid strategy for the construction of homogeneous label-free electrochemical DNA biosensors.

关 键 词：G-四聚体脱氧核酶 超微电极 氯化高铁血红素 PIK3CA循环肿瘤DNA 循环伏安法 

分 类 号：O657.11[理学—分析化学] TP212.3[理学—化学]