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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

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.
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biosensors-02-00405-f005: 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.

Mentions: Increase in bacterial concentration as measured by SYTO®-9 signal was linearly correlated with time (y = 8.0763x − 43.509, R² = 0.9802). One concern with a capture-based approach is that the device might “saturate” or fill with the target particles after a certain time, and fluorescent signal will cease to increase. Because the device did not saturate with captured bacteria at this high starting concentration, we can reasonably expect that it will likewise not saturate at much lower clinically and environmentally relevant bacterial loads and DEP concentration will provide the signal enhancement that enables the sensitivity of this method. Additionally, to confirm detection of bacteria at lower concentrations, a series of measurements were performed on a separate DEP device, successfully concentrating as few as ten cells per mL to a level detectable by SYTO®-9 fluorescence (Figure 5).


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)

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

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

biosensors-02-00405-f005: 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.
Mentions: Increase in bacterial concentration as measured by SYTO®-9 signal was linearly correlated with time (y = 8.0763x − 43.509, R² = 0.9802). One concern with a capture-based approach is that the device might “saturate” or fill with the target particles after a certain time, and fluorescent signal will cease to increase. Because the device did not saturate with captured bacteria at this high starting concentration, we can reasonably expect that it will likewise not saturate at much lower clinically and environmentally relevant bacterial loads and DEP concentration will provide the signal enhancement that enables the sensitivity of this method. Additionally, to confirm detection of bacteria at lower concentrations, a series of measurements were performed on a separate DEP device, successfully concentrating as few as ten cells per mL to a level detectable by SYTO®-9 fluorescence (Figure 5).

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