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

doi:10.7503/cjcu20170012

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

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. 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 Haojun^1,², GAO Wanlei^1,², JING Fengxiang¹, LIU Songsheng¹, ZHOU Hongbo¹, JIA Chunping^1,^*(), JIN Qinghui¹, ZHAO Jianlong¹

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/Abstract

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

中图分类号:

O655.9

TrendMD:

袁浩钧, 郜晚蕾, 景奉香, 刘松生, 周洪波, 贾春平, 金庆辉, 赵建龙. 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. 高等学校化学学报, 2017, 38(7): 1140.

YUAN Haojun, GAO Wanlei, JING Fengxiang, LIU Songsheng, ZHOU Hongbo, JIA Chunping, JIN Qinghui, ZHAO Jianlong. Novel Microfluidic Droplet Digital PCR Chip for High Sensitive Detection of Nucleic Acid^†. Chem. J. Chinese Universities, 2017, 38(7): 1140.

图/表 8

Fig.1 Design and operation of ddPCR device(A) Schematic representation of the ddPCR device; (B) structure of the PDMS chip(inset shows the generation of droplets under the microscope); (C) an image of the fabricated ddPCR chip.

Table 1 DNA sequences of primers and MGB probe

Sample	Sequence(5'—3')
Forward primer	atcccagaaggtgagaaagt
Reverse primer	tgtggagatgagcagggtct
MGB probe	HEX-aagccaacaaggaaatc-MGB; FAM-agatgttttgatagcgac-MGB

Fig.2 Bright-field images of the droplets after amplification in PDMS reservoir(A) and in glass reservoir of this ddPCR chip(B)

Fig.3 Plots of droplet diameter with various oil pressures(A) and water pressures(B) Error bars represent the standard deviation of droplet diameter.

Fig.4 Fluorescent images for simultaneous detection of EGFR gene19 exon with various

Fig.5 Measured value in the ddPCR assay against its expected valueError bars depict 95% confidence interval.

Fig.6 Detection of the EGFR exon19 rare mutant(0—0.01%) in the presence of wild type DNA by droplet digital PCR (A1—A4) Hex channel; (B1—B4) Fam channel. Rare mutant: (A1, B1) 0%; (A2, B2) 0.0001%; (A3, B3) 0.001%; (A4, B4) 0.01%.

Fig.7 Detection of the EGFR exon19 rare mutant(0.1%—100%)in the presence of wild type DNA by droplet digital PCR (A1—A4) Hex channel; (B1—B4) Fam channel. Rare mutant: (A1, B1) 0.1%; (A2, B2) 1%; (A3, B3) 10%; (A4, B4) 100%.

参考文献 33

[1]	Kalinina O., Lebedeva I., Brown J., Silver J., Nucleic Acids Research,1997, 25(10), 1999—2004
[2]	Vogelstein B., Kinzler K. W., Proceedings of the National Academy of Sciences of the United States of America,1999, 96(16), 9236—9241
[3]	Zhong Q., Bhattacharya S., Kotsopoulos S., Olson J., Taly V., Griffiths A. D., Link D. R., Larson J. W., Labona Chip,2011, 11(13), 2167—2174
[4]	Courtois F., Olguin L. F., Whyte G., Bratton D., Huck W. T. S., Abell C., Hollfelder F., Chem. Bio. Chem., 2008, 9(3), 439—446
[5]	Hatch A. C., Fisher J. S., Pentoney S. L., Yang D. L., Lee A. P., Labona Chip,2011, 11(13), 2509—2517
[6]	LermoA., Zacco E., Barak J., Delwiche M., Campoy S., Barbe J., Alegret S., Pividori M. I., >Biosensors & Bioelectronics,2008, 23(12), 1805—1811
[7]	Zhao Y. N., Petraitiene R., Walsh T. J., Perlin D. S.,J. Clinical Microbiology,2013, 51(3), 1034—1036
[8]	Higuchi R., Fockler C., Dollinger G., Watson R., Nature Biotechnology,1993, 11(9), 1026—1030
[9]	Yin J. L., Shackel N. A., Zekry A., Mcguinness P. H., Richards C., Putten K. V. D., Immunology & Cell Biology,2001, 79(3), 213—221
[10]	Blotta I., Prestinaci F., Mirante S., Cantafora A., Annali Dellistituto Superiore Di Sanita,2005, 41(1), 119—123
[11]	Mikael K. A., José M. A. B., Martin B. A., Ami F. D., Jiri J. E., Kristina L. A., Molecular Aspects of Medicine,2006, 27(27), 95—125
[12]	Houssin T., Cramer J., Grojsman R., Bellahsene L., Colas G., Moulet H., Lab on a Chip,2016, 16(8), 1401—1411
[13]	Moschou D., Vourdas N., Kokkoris G., Papadakis G., Parthenios J., Chatzandroulis S., Sensors & Actuators B: Chemical,2014, 199(4), 470—478
[14]	Hindson C. M., Chevillet J. R., Briggs H. A., Gallichotte E. N., Ruf I. K., Hindson B. J., Nature Methods,2013, 10(10), 1003
[15]	Dingle T. C., Sedlak R. H., Cook L., Jerome K. R., Clinical Chemistry,2013, 59(11), 1670—1672
[16]	Lynch T. J., Adjei A. A., Jr B. P., Eisen T. G., Engelman J., Goss G. D., Clinical Cancer Research,2006, 12(2), 4365s—4371s
[17]	Lynch T. J., Bell D. W., Sordella R., Gurubhagavatula S., Okimoto R. A., Brannigan B. W., New England Journal of Medicine,2004, 350(350), 2129—2139
[18]	Ciardiello F., Tortora G., New England Journal of Medicine,2008, 358(11), 1160—1174
[19]	Sharma S. V., Bell D. W., Settleman J., Haber D. A., Nature Reviews Cancer,2007, 7(3), 169—181
[20]	Rebecca C. H., Alissa G., Tinsley S., David L., Lynn H., Michelle F., Phil B., Jeanne H., Zheng L., PLoS One,2013, 8(8), e73068
[21]	Hatch A. C., Fisher J. S., Tovar A. R., Hsieh A. T., Lin R., Pentoney S. L., Lab on a Chip,2011, 11(22), 3838—3845
[22]	Courtois F., Olguin L. F., Whyte G., Bratton D., Huck W. T. S., Abell C., Chem. Bio. Chem., 2008, 9(3), 439—446
[23]	Taly V., Pekin D., El A. A., Laurent-Puig P., Trends in Molecular Medicine,2012, 18(7), 405—416
[24]	Ma J., Li N., Guarnera M., Feng J., Biomarker Insights,2013, 8(8), 127—136
[25]	Pornprasert S., Prasing W., European Journal of Haematology,2014, 92(3), 244—248
[26]	Thorsen T., Maerkl S. J., Quake S. R., Science,2002, 298(5593), 580—584
[27]	Dressman D., Yan H., Traverso G., Kinzler K. W., Vogelstein B. , Proc. Natl. Acad. Sci. USA,2003, 100(15), 8817—8822
[28]	Shigematsu H., Gazdar A. F., International Journal of Cancer,2006, 118(2), 257—262
[29]	Nakano M., Komatsu J. S., Takashima K., Katsura S., Mizuno A., J. Biotec., 2003, 2(2), 117—124
[30]	Bian X. J., Jing F. X., Li G., Biosensors and Bioelectronics,2015, 74, 770—777
[31]	Mazutis L., Araghi A. F., Miller O. J., Baret J. C., Frenz L., Janoshazi A., Analy. Chem., 2009, 81(12), 4813—4821
[32]	Heyries K. A., Tropini C., Vaninsberghe M., Doolin C., Petriv O. I., Singhal A., Nature Methods,2011, 8(8), 649—651
[33]	Kiss M. M., Ortoleva-Donnelly L., Beer N. R., Warner J., Bailey C. G., Colston B. W., Rothberg J. M., Link D. R., Leamon J. H. , Anal. Chem., 2008, 80(23), 8975—8981

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

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

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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

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

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