高等学校化学学报 ›› 2020, Vol. 41 ›› Issue (8): 1760-1767.doi: 10.7503/cjcu20200308

• 分析化学 • 上一篇    下一篇

一种用于核酸绝对定量检测的高鲁棒性液滴式数字PCR芯片

彭伙1,2, 高则航1,2,3, 廖承悦1,2, 王晓冬4, 周洪波1, 赵建龙1   

  1. 1. 中国科学院上海微系统与信息技术研究所, 传感技术联合国家重点实验室, 上海 200050;
    2. 中国科学院大学, 北京 100049;
    3. 上海科技大学信息科学与技术学院, 上海 201210;
    4. 北京旌微医学工程研究院有限公司, 北京 100176
  • 收稿日期:2020-05-29 出版日期:2020-08-10 发布日期:2020-08-01
  • 通讯作者: 周洪波,男,博士,副研究员,主要从事微纳生化传感器方面的研究.E-mail:zhouhb@mail.sim.ac.cn;赵建龙,男,博士,研究员,主要从事生物芯片和生物传感器方面的研究.E-mail:jlzhao@mail.sim.ac.cn E-mail:zhouhb@mail.sim.ac.cn;jlzhao@mail.sim.ac.cn
  • 基金资助:
    国家重点研发计划项目(批准号:2017YFA0205303,2017ZX10302201005006)、中国科学院青年创新促进会(批准号:2018267)、中国科学院科研装备研究项目(批准号:YJKYYQ20170043)、上海市科学技术委员会科技支撑计划(批准号:17441904300)、国家自然科学基金(批准号:61804158)和上海市青年科技英才扬帆计划(批准号:18YF1428300)资助.

Robust Droplet Digital PCR Chip for Absolute Quantitative Detection of Nucleic Acid

PENG Huo1,2, GAO Zehang1,2,3, LIAO Chengyue1,2, WANG Xiaodong4, ZHOU Hongbo1, 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;
    3. School of Information Science and Technology, Shanghai Tech University, Shanghai 201210, China;
    4. Beijing Genome Way Medical Engineering Research Institute Co., Ltd., Beijing 100176, China
  • Received:2020-05-29 Online:2020-08-10 Published:2020-08-01
  • Supported by:
    Supported by the National Key Research and Development Program of China(Nos.2017YFA0205303, 2017ZX10302201005006), the Youth Innovation Promotion Association, CAS(No.2018267), the Research Project of Scientific Research Equipment of CAS(No.YJKYYQ20170043), the Science and Technology Support Plan of the Shanghai Science and Technology Committee, China(No.17441904300), the National Natural Science Foundation of China(No.61804158) and the Shanghai Sailing Program, China(No.18YF1428300).

摘要: 为满足液滴式数字聚合酶链式反应(PCR)技术对扩增反应过程中稳定保存液滴以及反应后高效检测的核心需求,构建了一种具有过滤气泡和增强荧光信号功能的液滴式数字聚合酶链式反应芯片.该芯片可在10 min内产生20多万个半径约为21 μm的液滴.利用“玻璃天花板”的方式构建了独立于芯片主体材料的液滴收集腔,为液滴提供稳定的保存与反应环境;还构建了过滤结构,可有效过滤混入液相中的空气,提高芯片鲁棒性.同时,在液滴收集腔中引入反射层,增强荧光信号,使单个视野荧光成像时间缩短约40%,提高了检测效率.利用该芯片定量检测EGFR基因第21号外显子,检测信号与DNA浓度在101~105 copies/μL范围内呈现良好的线性关系(R2=0.998).该方案在载玻片大小的芯片上实现了液滴产生、PCR扩增和荧光信号读取,并具有较高的鲁棒性与检测效率,在核酸检测等方面具有应用潜力.

关键词: 数字聚合酶链式反应, 液滴微流控芯片, 核酸定量检测, 脱氧核糖核酸

Abstract: A highly robust droplet digital polymerase chain reaction(ddPCR) chip with filtering bubbles and enhancing fluorescence is constructed to meet the core requirements of the stable preservation of droplets in the amplification process and the high-efficiency detection after the reaction. The chip could produce more than 2×105 droplets with a radius of ca. 21 μm in 10 min. The droplet collection chamberis built independent of the chip material via "glass ceiling" method, provided a stable environment for droplets. At the same time, the filter structure is constructed to effectively filter the air mixed into the liquid phase, improving the robustness of the chip. The reflector, introduced into the collecting chamber, enhances the fluorescence signal, reducing exposure time and improving the detection efficiency. Using this chip to detect EGFR exon 21, a good linear correlation with the DNA concentration from 101 to 105 copies/μL(R2=0.998) could be obtained. In general, the scheme, which achieved droplet generation, PCR amplification and fluorescence readout on the chip robustly and efficiently, has potential in nucleic acid detection and related applications.

Key words: Digital polymerase chain reaction(PCR), Droplet microfluidic chip, Nucleic acid detection, DNA

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