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Biosensors 2012, 2(4), 405-416; doi:10.3390/bios2040405
Article

Microfluidic-Based Amplification-Free Bacterial DNA Detection by Dielectrophoretic Concentration and Fluorescent Resonance Energy Transfer Assisted in Situ Hybridization (FRET-ISH)

1,2,#, 2
 and 1,*
1 Nanobiosensors Laboratory, Michigan State University, East Lansing, MI 48824, USA 2 Lawrence Livermore National Laboratory, 7000 East Ave., Livermore, CA 94550, USA # This author deceased on 4 July 2012.
This work performed partly under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. LLNL-JRNL-561017; Based on “Packard, M.M.; Shusteff, M.; Alocilja, E.C. Novel, rapid DNA-based on-chip bacterial identification system combining dielectrophoresis and amplification-free fluorescent resonance energy transfer assisted in-situ hybridization (FRET-ISH). Proc. SPIE 2011, doi: 10.1117/12.892425.
* Author to whom correspondence should be addressed.
Received: 20 August 2012 / Revised: 24 September 2012 / Accepted: 8 October 2012 / Published: 10 October 2012
(This article belongs to the Special Issue Nano and Micro DNA/RNA Sensors)
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Abstract

Although real-time PCR (RT-PCR) has become a diagnostic standard for rapid identification of bacterial species, typical methods remain time-intensive due to sample preparation and amplification cycle times. The assay described in this work incorporates on-chip dielectrophoretic capture and concentration of bacterial cells, thermal lysis, cell permeabilization, and nucleic acid denaturation and fluorescence resonance energy transfer assisted in situ hybridization (FRET-ISH) species identification. Combining these techniques leverages the benefits of all of them, allowing identification to be accomplished completely on chip less than thirty minutes after receipt of sample, compared to multiple hours required by traditional RT-PCR and its requisite sample preparation.
Keywords: fluorescence resonance energy transfer assisted in situ hybridization (FRET-ISH); in situ hybridization (ISH); dielectrophoresis (DEP); on-chip diagnostics; lab on a chip; microbial identification fluorescence resonance energy transfer assisted in situ hybridization (FRET-ISH); in situ hybridization (ISH); dielectrophoresis (DEP); on-chip diagnostics; lab on a chip; microbial identification
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Packard, M.M.; Shusteff, M.; Alocilja, E.C. Microfluidic-Based Amplification-Free Bacterial DNA Detection by Dielectrophoretic Concentration and Fluorescent Resonance Energy Transfer Assisted in Situ Hybridization (FRET-ISH). Biosensors 2012, 2, 405-416.

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