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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

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.
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biosensors-02-00405-f001: 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.

Mentions: Dielectrophoresis was performed inside silicon-and-glass chips fabricated using standard cleanroom microfabrication techniques. In brief, a 4'' silicon wafer was first wet-oxidized to form a 200 nm SiO2 insulating layer, on top of which 250 nm of Cr-Au metal was sputter-deposited. The metal was patterned by standard photolithography and wet-etching (AZ 1518 resist, Transene gold etch TFA, Cyantek CR-7 chrome etchant). A second 4'' wafer made of borosilicate glass was drilled with 500 µm diameter through-holes (Bullen Ultrasonics) to provide fluid access ports. After drilling, a Cr-Au metal layer was sputter-deposited to serve as a mask for fluid channel etching. The fluid channel pattern was wet-etched in the metal mask, and then the glass was etched to a depth of 10–15 µm using a solution of 22% hydrofluoric acid and 78% acetic acid. After stripping the metal etch-mask, the glass and silicon chips were anodically bonded together (350 °C, constant voltage −900 V, ~5 min) to form sealed fluid channels 2.6 mm wide and 60 mm long. Interdigitated electrodes in the chips (Figure 1) were 40 µm wide with 40 µm spacing. Individual chips were diced apart and wire leads were attached using silver paint and epoxy to electrode contact pads connected to each side of the interdigitated electrode array. Serial dilutions of bacteria stained only with SYTO®-9 were flowed at 100 µL/min for one minute and dielectrophoretically captured and concentrated at a frequency of 1 MHz and voltage of 10 Vp-p supplied by a standard digital function and waveform generator (Agilent 33220 A) directly to the chip leads.


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)

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

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

biosensors-02-00405-f001: 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.
Mentions: Dielectrophoresis was performed inside silicon-and-glass chips fabricated using standard cleanroom microfabrication techniques. In brief, a 4'' silicon wafer was first wet-oxidized to form a 200 nm SiO2 insulating layer, on top of which 250 nm of Cr-Au metal was sputter-deposited. The metal was patterned by standard photolithography and wet-etching (AZ 1518 resist, Transene gold etch TFA, Cyantek CR-7 chrome etchant). A second 4'' wafer made of borosilicate glass was drilled with 500 µm diameter through-holes (Bullen Ultrasonics) to provide fluid access ports. After drilling, a Cr-Au metal layer was sputter-deposited to serve as a mask for fluid channel etching. The fluid channel pattern was wet-etched in the metal mask, and then the glass was etched to a depth of 10–15 µm using a solution of 22% hydrofluoric acid and 78% acetic acid. After stripping the metal etch-mask, the glass and silicon chips were anodically bonded together (350 °C, constant voltage −900 V, ~5 min) to form sealed fluid channels 2.6 mm wide and 60 mm long. Interdigitated electrodes in the chips (Figure 1) were 40 µm wide with 40 µm spacing. Individual chips were diced apart and wire leads were attached using silver paint and epoxy to electrode contact pads connected to each side of the interdigitated electrode array. Serial dilutions of bacteria stained only with SYTO®-9 were flowed at 100 µL/min for one minute and dielectrophoretically captured and concentrated at a frequency of 1 MHz and voltage of 10 Vp-p supplied by a standard digital function and waveform generator (Agilent 33220 A) directly to the chip leads.

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