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

Packard MM, Shusteff M, Alocilja EC - Biosensors (Basel) (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Nanobiosensors Laboratory, Michigan State University, East Lansing, MI 48824, USA.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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biosensors-02-00405-f004: 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.

Mentions: Whereas the initial on-chip cell population is barely detectable at a starting concentration of 106 cfu/mL (Figure 3(a)), bacterial presence at the electrodes after concentration is evident and easily discernible (Figure 3(b)). Flowing at a rate of 100 µL/min for one minute, bacteria were successfully captured and concentrated greater than 400 times by dielectrophoresis (Figure 3 and Figure 4).


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

Packard MM, Shusteff M, Alocilja EC - Biosensors (Basel) (2012)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263560&req=5

biosensors-02-00405-f004: 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.
Mentions: Whereas the initial on-chip cell population is barely detectable at a starting concentration of 106 cfu/mL (Figure 3(a)), bacterial presence at the electrodes after concentration is evident and easily discernible (Figure 3(b)). Flowing at a rate of 100 µL/min for one minute, bacteria were successfully captured and concentrated greater than 400 times by dielectrophoresis (Figure 3 and Figure 4).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Nanobiosensors Laboratory, Michigan State University, East Lansing, MI 48824, USA.

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.

No MeSH data available.


Related in: MedlinePlus