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A Simple, Valveless Microfluidic Sample Preparation Device for Extraction and Amplification of DNA from Nanoliter-Volume Samples

Lindsay A. Legendre,^†^‡ Joan M. Bienvenue,^†^‡ Michael G. Roper,^† Jerome P. Ferrance,^† and James P. Landers*^†^§

Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, and Department of Pathology, University of Virginia Health Science Center, Charlottesville, Virginia 22908

Anal. Chem., 2006, 78 (5), pp 1444–1451

DOI: 10.1021/ac0516988

Publication Date (Web): January 31, 2006

†

University of Virginia.

‡

These authors contributed equally to this work.

To whom correspondence should be addressed. Phone: (434)-243-8658. Fax: (434)-924-3048. E-mail: landers@virginia.edu.

University of Virginia Health Science Center.

Abstract

A glass microdevice has been constructed for the on-line integration of solid-phase extraction (SPE) of DNA and polymerase chain reaction (PCR) on a single chip. The chromatography required for SPE in the microfluidic sample preparation device (μSPD) was carried out in a silica bead/sol−gel SPE bed, where the purified DNA was eluted directly into a downstream chamber where conventional thermocycling allowed for PCR amplification of specific DNA target sequences. Through rapid, simple passivation of the PCR chamber with a silanizing reagent, reproducible DNA extraction and amplification was demonstrated from complex biological matrixes in a manner amenable to any research laboratory, using only a syringe pump and a conventional thermocycler. The μSPD allowed for SPE concentration of DNA from 600 nL of blood coupled to subsequent on-chip amplification that yielded a detectable amplicon; this simple device can be applied to a variety of routine genetic analyses without the need for sophisticated instrumentation. In addition, the applicability of these developments to nonconventional thermocycling was demonstrated through the use of noncontact, IR-mediated heating. This was exemplified with the isolation of DNA from an anthrax spore-spiked nasal swab and the subsequent on-chip amplification of target DNA sequences in a total processing time of only 25 min.

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