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. 2012 Oct 10;2(4):405-16.
doi: 10.3390/bios2040405.

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

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Free PMC article

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

Michelle M Packard et al. Biosensors (Basel). .
Free PMC article

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.

Figures

Figure 1
Figure 1
Dielectrophoresis chip design (a) top view and (b) cross-sectional view. Note that the fluid channel spans only the interdigitated portion of the electrodes, and the metal regions common to each set of electrodes do not come in contact with the fluid.
Figure 2
Figure 2
Quantitative Spectrofluorometry. Correlation of bacterial concentration and background subtracted (background = HEX probe signal with no bacterial DNA or donor dye present) FRET-ISH signal (acceptor dye emission at 560 nm) as recorded by the Nanodrop 3300.
Figure 3
Figure 3
Dielectrophoretic capture and concentration of bacterial cells. Both images have identical camera gain and contrast settings and identical scale/magnification. (a) Prior to dielectrophoretic capture and concentration, SYTO®-9 stained bacteria (106 cfu/mL) are barely detectable. (b) After one minute of capture at 1 MHz and 100 µL/min, bacteria are evident and signal intensity is over 400× greater.
Figure 4
Figure 4
Dielectrophoretic bacterial concentration. While sample solution flows past the electrodes, the total donor fluorescence signal, summed over the entire image, from SYTO®-9 labeled bacteria (no acceptor is present) increases linearly over time, measuring more than 400× (458.5 ± 18.2×, n = 6) the initial value after one minute flow at 100 µL/min.
Figure 5
Figure 5
Concentration of serial bacterial dilutions after sixty seconds of dielectrophoresis (DEP). Signal increases with initial bacterial concentration in the range of 101–106 cfu/mL.
Figure 6
Figure 6
Donor Photobleaching. Photobleaching of donor signal at 505 nm without acceptor (formula image) was significantly greater than donor in the presence of acceptor (- - -) when excited at 485/20 nm for 60 s.

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