A fully integrated microfluidic genetic analysis system with sample-in–answer-out capability

doi:10.1073/pnas.0604663103

A fully integrated microfluidic genetic analysis system with sample-in–answer-out capability

*Department of Chemistry, University of Virginia, Charlottesville, VA 22904;
Departments of ^†Comparative Medicine,
^‡Infectious Disease, and
^¶Pathology, University of Virginia Health System, Charlottesville, VA 22908; and
^§Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, MD 28092

Edited by Robert H. Austin, Princeton University, Princeton, NJ, and approved October 16, 2006 (received for review June 5, 2006)

Abstract

We describe a microfluidic genetic analysis system that represents a previously undescribed integrated microfluidic device capable of accepting whole blood as a crude biological sample with the endpoint generation of a genetic profile. Upon loading the sample, the glass microfluidic genetic analysis system device carries out on-chip DNA purification and PCR-based amplification, followed by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with sample-in–answer-out results in <30 min. A single syringe pump delivers sample/reagents to the chip for nucleic acid purification from a biological sample. Elastomeric membrane valving isolates each distinct functional region of the device and, together with resistive flow, directs purified DNA and PCR reagents from the extraction domain into a 550-nl chamber for rapid target sequence PCR amplification. Repeated pressure-based injections of nanoliter aliquots of amplicon (along with the DNA sizing standard) allow electrophoretic separation and detection to provide DNA fragment size information. The presence of Bacillus anthracis (anthrax) in 750 nl of whole blood from living asymptomatic infected mice and of Bordetella pertussis in 1 μl of nasal aspirate from a patient suspected of having whooping cough are confirmed by the resultant genetic profile.

Footnotes

^‖To whom correspondence should be addressed. E-mail: landers{at}virginia.edu
Author contributions: C.J.E., J.M.K., J.M.B., and L.A.L. contributed equally to this work; C.J.E., J.M.K., J.M.B., L.A.L., J.P.F., and J.P.L. designed research; C.J.E., J.M.K., J.M.B., and L.A.L. performed research; S.H.F., M.A.H., E.L.H., and T.J.M. contributed new reagents/analytic tools; C.J.E., J.M.K., J.M.B., L.A.L., M.G.R., and J.P.L. analyzed data; and C.J.E., J.M.K., J.M.B., L.A.L., M.G.R., M.A.H., and J.P.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
This article contains supporting information (SI) online at www.pnas.org/cgi/content/full/0604663103/DC1.
Abbreviations:

μ-TAS,

micrototal analysis system;

MGA,

microfluidic genetic analysis;

SPE,

solid-phase extraction;

ME,

microchip electrophoretic;

qPCR,

quantitative PCR;

PDMS,

poly(dimethyl siloxane).