Droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulations

Persistent Link:
http://hdl.handle.net/10150/610171
Title:
Droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulations
Author:
You, David; Yoon, Jeong-Yeol
Affiliation:
Department of Agricultural and Biosystems Engineering, The University of Arizona, Tucson, AZ 85721-0038, USA
Issue Date:
2012
Publisher:
BioMed Central
Citation:
You and Yoon Journal of Biological Engineering 2012, 6:15 http://www.jbioleng.org/content/6/1/15
Journal:
Journal of Biological Engineering
Rights:
© 2012 You and Yoon; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0)
Collection Information:
This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at repository@u.library.arizona.edu.
Abstract:
A computer numerical control (CNC) apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using "wire-guided" method (a pipette tip was used in this study). This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate). Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 muL droplet at 2300 round per minute, concentrating E. coli by more than 3-fold within 3min. DNA extraction was demonstrated from E. coli sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10min. Following extraction, the 1500bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10min for 30cycles. The total assay time was 23min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 muL droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5min (during DNA extraction). The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.
EISSN:
1754-1611
DOI:
10.1186/1754-1611-6-15
Keywords:
Droplet manipulations; Escherichia coli; Peptidase D; Droplet PCR; Rapid PCR
Version:
Final published version
Additional Links:
http://www.jbioleng.org/content/6/1/15

Full metadata record

DC FieldValue Language
dc.contributor.authorYou, Daviden
dc.contributor.authorYoon, Jeong-Yeolen
dc.date.accessioned2016-05-20T09:00:15Z-
dc.date.available2016-05-20T09:00:15Z-
dc.date.issued2012en
dc.identifier.citationYou and Yoon Journal of Biological Engineering 2012, 6:15 http://www.jbioleng.org/content/6/1/15en
dc.identifier.doi10.1186/1754-1611-6-15en
dc.identifier.urihttp://hdl.handle.net/10150/610171-
dc.description.abstractA computer numerical control (CNC) apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using "wire-guided" method (a pipette tip was used in this study). This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate). Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 muL droplet at 2300 round per minute, concentrating E. coli by more than 3-fold within 3min. DNA extraction was demonstrated from E. coli sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10min. Following extraction, the 1500bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10min for 30cycles. The total assay time was 23min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 muL droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5min (during DNA extraction). The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.en
dc.language.isoenen
dc.publisherBioMed Centralen
dc.relation.urlhttp://www.jbioleng.org/content/6/1/15en
dc.rights© 2012 You and Yoon; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0)en
dc.subjectDroplet manipulationsen
dc.subjectEscherichia colien
dc.subjectPeptidase Den
dc.subjectDroplet PCRen
dc.subjectRapid PCRen
dc.titleDroplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulationsen
dc.typeArticleen
dc.identifier.eissn1754-1611en
dc.contributor.departmentDepartment of Agricultural and Biosystems Engineering, The University of Arizona, Tucson, AZ 85721-0038, USAen
dc.identifier.journalJournal of Biological Engineeringen
dc.description.collectioninformationThis item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at repository@u.library.arizona.edu.en
dc.eprint.versionFinal published versionen
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