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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

Donor Photobleaching. Photobleaching of donor signal at 505 nm without acceptor () was significantly greater than donor in the presence of acceptor (- - -) when excited at 485/20 nm for 60 s.
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biosensors-02-00405-f006: Donor Photobleaching. Photobleaching of donor signal at 505 nm without acceptor () was significantly greater than donor in the presence of acceptor (- - -) when excited at 485/20 nm for 60 s.

Mentions: FRET-ISH efficiency was determined by quantification of donor dye photobleaching (Figure 6). A decaying exponential fitted to relative intensity over time found photobleaching decay time constant of donor dye alone (τpb) and in the presence of the HEX-labeled ERIC probe (τ'pb) to be 31.8 s and 135.1 s, respectively (Table 2). Photobleaching decay time constants of emission at 505 nm were calculated for SYTO®-9 stained bacterial samples unbound and bound to the HEX-labeled ERIC probe. The FRET efficiency, E, was then determined to be 76.4% (Table 2), indicative of exceptional probe binding within nanoscale proximity of the SYTO®-9 dye.


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)

Donor Photobleaching. Photobleaching of donor signal at 505 nm without acceptor () was significantly greater than donor in the presence of acceptor (- - -) when excited at 485/20 nm for 60 s.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

biosensors-02-00405-f006: Donor Photobleaching. Photobleaching of donor signal at 505 nm without acceptor () was significantly greater than donor in the presence of acceptor (- - -) when excited at 485/20 nm for 60 s.
Mentions: FRET-ISH efficiency was determined by quantification of donor dye photobleaching (Figure 6). A decaying exponential fitted to relative intensity over time found photobleaching decay time constant of donor dye alone (τpb) and in the presence of the HEX-labeled ERIC probe (τ'pb) to be 31.8 s and 135.1 s, respectively (Table 2). Photobleaching decay time constants of emission at 505 nm were calculated for SYTO®-9 stained bacterial samples unbound and bound to the HEX-labeled ERIC probe. The FRET efficiency, E, was then determined to be 76.4% (Table 2), indicative of exceptional probe binding within nanoscale proximity of the SYTO®-9 dye.

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