Droplet microfluidics for amplification-free genetic detection of single cells

被引：75

作者：

Rane, Tushar D. ^{[1
]}

Zec, Helena C. ^{[1
]}

Puleo, Chris ^{[1
]}

Lee, Abraham P. ^{[2
]}

Wang, Tza-Huei ^{[1
,3
]}

机构：

[1] Johns Hopkins Univ, Dept Biomed Engn, Baltimore, MD 21218 USA

[2] Univ Calif Irvine, Dept Biomed Engn, Irvine, CA USA

[3] Johns Hopkins Univ, Dept Mech Engn, Baltimore, MD 21218 USA

来源：

LAB ON A CHIP | 2012年 / 12卷 / 18期

基金：

美国国家科学基金会;

关键词：

RIBOSOMAL-RNA GENE; DIGITAL PCR; MOLECULAR BEACONS; QUANTITATIVE DETECTION; NUCLEIC-ACIDS; 16S; BACTERIA; DNA; QUANTIFICATION; HYBRIDIZATION;

D O I：

10.1039/c2lc40537g

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

In this article we present a novel droplet microfluidic chip enabling amplification-free detection of single pathogenic cells. The device streamlines multiple functionalities to carry out sample digitization, cell lysis, probe-target hybridization for subsequent fluorescent detection. A peptide nucleic acid fluorescence resonance energy transfer probe (PNA beacon) is used to detect 16S rRNA present in pathogenic cells. Initially the sensitivity and quantification abilities of the platform are tested using a synthetic target mimicking the actual expression level of 16S rRNA in single cells. The capability of the device to perform "sample-to-answer'' pathogen detection of single cells is demonstrated using E. coli as a model pathogen.

引用

页码：3341 / 3347

页数：7

共 30 条

[1] Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity [J].

Baret, Jean-Christophe ;

Miller, Oliver J. ;

Taly, Valerie ;

Ryckelynck, Michael ;

El-Harrak, Abdeslam ;

Frenz, Lucas ;

Rick, Christian ;

Samuels, Michael L. ;

Hutchison, J. Brian ;

Agresti, Jeremy J. ;

Link, Darren R. ;

Weitz, David A. ;

Griffiths, Andrew D. .

LAB ON A CHIP, 2009, 9 (13) :1850-1858

[2] Detecting bacteria and determining their susceptibility to antibiotics by stochastic confinement in nanoliter droplets using plug-based microfluidics [J].

Boedicker, James Q. ;

Li, Liang ;

Kline, Timothy R. ;

Ismagilov, Rustem F. .

LAB ON A CHIP, 2008, 8 (08) :1265-1272

[3]

BROSIUS J, 1978, P NATL ACAD SCI USA, V75, P4801, DOI 10.1073/pnas.75.10.4801

[4] Droplet microfluidic technology for single-cell high-throughput screening [J].

Brouzes, Eric ;

Medkova, Martina ;

Savenelli, Neal ;

Marran, Dave ;

Twardowski, Mariusz ;

Hutchison, J. Brian ;

Rothberg, Jonathan M. ;

Link, Darren R. ;

Perrimon, Norbert ;

Samuels, Michael L. .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2009, 106 (34) :14195-14200

[5] Droplet-based microfluidic platforms for the encapsulation and screening of mammalian cells and multicellular organisms [J].

Clausell-Tormos, Jenifer ;

Lieber, Diana ;

Baret, Jean-Christophe ;

El-Harrak, Abdeslam ;

Miller, Oliver J. ;

Frenz, Lucas ;

Blouwolff, Joshua ;

Humphry, Katherine J. ;

Koster, Sarah ;

Duan, Honey ;

Holtze, Christian ;

Weitz, David A. ;

Griffiths, Andrew D. ;

Merten, Christoph A. .

CHEMISTRY & BIOLOGY, 2008, 15 (05) :427-437

[6] Widespread archaea and novel bacteria from the deep sea as shown by 16S rRNA gene sequences [J].

Fuhrman, JA ;

Davis, AA .

MARINE ECOLOGY PROGRESS SERIES, 1997, 150 (1-3) :275-285

[7] High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number [J].

Hindson, Benjamin J. ;

Ness, Kevin D. ;

Masquelier, Donald A. ;

Belgrader, Phillip ;

Heredia, Nicholas J. ;

Makarewicz, Anthony J. ;

Bright, Isaac J. ;

Lucero, Michael Y. ;

Hiddessen, Amy L. ;

Legler, Tina C. ;

Kitano, Tyler K. ;

Hodel, Michael R. ;

Petersen, Jonathan F. ;

Wyatt, Paul W. ;

Steenblock, Erin R. ;

Shah, Pallavi H. ;

Bousse, Luc J. ;

Troup, Camille B. ;

Mellen, Jeffrey C. ;

Wittmann, Dean K. ;

Erndt, Nicholas G. ;

Cauley, Thomas H. ;

Koehler, Ryan T. ;

So, Austin P. ;

Dube, Simant ;

Rose, Klint A. ;

Montesclaros, Luz ;

Wang, Shenglong ;

Stumbo, David P. ;

Hodges, Shawn P. ;

Romine, Steven ;

Milanovich, Fred P. ;

White, Helen E. ;

Regan, John F. ;

Karlin-Neumann, George A. ;

Hindson, Christopher M. ;

Saxonov, Serge ;

Colston, Bill W. .

ANALYTICAL CHEMISTRY, 2011, 83 (22) :8604-8610

[8] Quantitative detection of protein expression in single cells using droplet microfluidics [J].

Huebner, A. ;

Srisa-Art, M. ;

Holt, D. ;

Abell, C. ;

Hollfelder, F. ;

deMello, A. J. ;

Edel, J. B. .

CHEMICAL COMMUNICATIONS, 2007, (12) :1218-1220

[9] Detection and Analysis of Low-Abundance Cell-Surface Biomarkers Using Enzymatic Amplification in Microfluidic Droplets [J].

Joensson, Haakan N. ;

Samuels, Michael L. ;

Brouzes, Eric R. ;

Medkova, Martina ;

Uhlen, Mathias ;

Link, Darren R. ;

Andersson-Svahn, Helene .

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 2009, 48 (14) :2518-2521

[10] Drop-based microfluidic devices for encapsulation of single cells [J].

Koester, Sarah ;

Angile, Francesco E. ;

Duan, Honey ;

Agresti, Jeremy J. ;

Wintner, Anton ;

Schmitz, Christian ;

Rowat, Amy C. ;

Merten, Christoph A. ;

Pisignano, Dario ;

Griffiths, Andrew D. ;

Weitz, David A. .

LAB ON A CHIP, 2008, 8 (07) :1110-1115

← 1 2 3 →