高等学校化学学报 ›› 2017, Vol. 38 ›› Issue (7): 1140-1147.doi: 10.7503/cjcu20170012

• 分析化学 • 上一篇    下一篇

一种用于核酸高灵敏检测的液滴式数字聚合酶链式反应芯片

袁浩钧1,2, 郜晚蕾1,2, 景奉香1, 刘松生1, 周洪波1, 贾春平1(), 金庆辉1, 赵建龙1   

  1. 1. 传感技术国家重点实验室, 中国科学院上海微系统与信息技术研究所, 上海 200050
    2. 中国科学院大学, 北京 100049
  • 收稿日期:2017-01-09 出版日期:2017-07-10 发布日期:2017-06-12
  • 作者简介:联系人简介: 贾春平, 女, 博士, 副研究员, 主要从事纳米生物传感器、 生物芯片、 肿瘤检测及微系统分析仪等方面的研究. E-mail: jiachp@mail.sim.ac.cn
  • 基金资助:
    国家自然科学基金(批准号: 81472751, 61271162, 61571428, 61571429)、 国家“九七三”计划项目(批准号: 2012CB933303) 和 上海市浦江人才计划项目(批准号: 15PJ1409800)资助

Novel Microfluidic Droplet Digital PCR Chip for High Sensitive Detection of Nucleic Acid

YUAN Haojun1,2, GAO Wanlei1,2, JING Fengxiang1, LIU Songsheng1, ZHOU Hongbo1, JIA Chunping1,*(), JIN Qinghui1, ZHAO Jianlong1   

  1. 1. State Key Laboratory of Transducer Technology,Shanghai Institute of Microsystem and Information Technology,Chinese Academy of Sciences, Shanghai 200050, China
    2. University of Chinese Academy of Sciences, Beijing 100049, China
  • Received:2017-01-09 Online:2017-07-10 Published:2017-06-12
  • Contact: JIA Chunping E-mail:jiachp@mail.sim.ac.cn
  • Supported by:
    † Supported by the National Natural Science Foundation of China(Nos.81472751, 61271162, 61571428, 61571429), the National Basic Research Program of China(No.2012CB933303) and the Shanghai Pujiang Program of China(No.15PJ1409800)

摘要:

为了实现对核酸的高灵敏度检测, 构建了一种新型的液滴式数字聚合酶链式反应(ddPCR)芯片. 该芯片由产生液滴的聚二甲基硅氧烷(PDMS)模块和储存液滴的玻璃腔室构成. 实验结果表明, 该芯片可以在25 min内产生2×106个直径为20 μm的微液滴(体积4.187 pL). 利用该芯片定量检测了表皮生长因子受体(EGFR)基因第19号外显子, 在DNA浓度为106~101 copies/μL范围内呈现良好的线性关系(R2=0.9998); 在浓度为106 copies/μL的19号外显子野生型DNA中检测105~100 copies/μL的突变型DNA, 其检测敏感度可达到0.0001%. 该方法在同一芯片上实现了液滴产生、 核酸扩增和荧光信号读取的功能, 在核酸绝对定量及痕量突变基因的检测中具有潜在应用前景.

关键词: 数字聚合酶链式反应, 微流控芯片, 微液滴, 高灵敏度核酸检测

Abstract:

Sensitive and rapid quantification of nucleic acid is significant for clinical diagnosis and medication guide of tumor. In this study, we developed a novel droplet digital PCR(ddPCR) device for sensitive nucleic acid detection. This chip contains a PDMS chip and glass-made reservoir, which can overcome the problems of droplet evaporation and instability during PCR. The double droplet generation channels in this chip can produce 2×106 droplets(4.187 pL each) in 25 min. For detecting EGFR exon 19 wild-type DNA using this ddPCR chip, it demonstrated a good linear correlation with the DNA concentration from 10 to 106 copies/μL(R2=0.9998). In addition, we show sensitive detection of EGFR exon 19 mutate DNA in a 106-fold excess of wild-type background. In conclusion, this ddPCR chip integrates the functions of droplet generation, on-chip PCR process and fluorescence signals readout, which will provide a potential application in the absolute nucleic acids quantification and rare mutate gene detection.

Key words: Digital PCR, Microfluidic chip, Micro-droplet, High sensitive nucleic acid detection

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