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Propidium monoazide pretreatment on a 3D-printed microfluidic device for efficient PCR determination of 'live versus dead' microbial cells

Zhu, Yanzhe and Huang, Xiao and Xie, Xing and Bahnemann, Janina and Lin, Xingyu and Wu, Xunyi and Wang, Siwen and Hoffmann, Michael R. (2018) Propidium monoazide pretreatment on a 3D-printed microfluidic device for efficient PCR determination of 'live versus dead' microbial cells. Environmental Science: Water Research & Technology, 4 (7). pp. 956-963. ISSN 2053-1400. https://resolver.caltech.edu/CaltechAUTHORS:20180611-134345584

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Abstract

Waterborne microbial pathogen detection via nucleic acid analysis on portable microfluidic devices is a growing area of research, development, and application. Traditional polymerase chain reaction (PCR)-based nucleic acid analysis detects total extracted DNA, but cannot differentiate live and dead cells. A propidium monoazide (PMA) pretreatment step before PCR can effectively exclude DNA from nonviable cells, as PMA can selectively diffuse through compromised cell membranes and intercalate with DNA to form DNA–PMA complex upon light exposure. The complex strongly inhibits the amplification of the bound DNA in PCR, and thus, only cells with intact cell membranes are detected. Herein, this study reports the development of a microfluidic device to carry out PMA pretreatment ‘on-chip’. Chip design was guided by computer simulations, and prototypes were fabricated using a high-resolution 3D printer. The optimized design utilizes split and recombine mixers for initial PMA-sample mixing and a serpentine flow channel containing herringbone structures for dark and light incubation. On-chip PMA pretreatment to differentiate live and dead bacterial cells in buffer and natural pond water samples was successfully demonstrated.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1039/c8ew00058aDOIArticle
http://www.rsc.org/suppdata/c8/ew/c8ew00058a/c8ew00058a1.pdfPublisherSupplementary Information
ORCID:
AuthorORCID
Zhu, Yanzhe0000-0002-2260-1830
Hoffmann, Michael R.0000-0002-0432-6564
Additional Information:© 2018 The Royal Society of Chemistry. The article was received on 31 Jan 2018, accepted on 11 Apr 2018 and first published on 11 Jun 2018. The authors acknowledge the financial support of the Bill and Melinda Gates Foundation (BMGF OPP1111246). Conflicts of interest: There are no conflicts to declare.
Funders:
Funding AgencyGrant Number
Bill and Melinda Gates FoundationOPP1111246
Issue or Number:7
Record Number:CaltechAUTHORS:20180611-134345584
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180611-134345584
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:86966
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:11 Jun 2018 20:54
Last Modified:03 Oct 2019 19:50

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