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A DNA biochip for on-the-spot multiplexed pathogen identification.

Yeung SW, Lee TM, Cai H, Hsing IM - Nucleic Acids Res. (2006)

Bottom Line: Oligonucleotide probes specific to the target amplicons are individually positioned at each ITO surface by electrochemical copolymerization of pyrrole and pyrrole-probe conjugate.These immobilized probes were stable to the thermal cycling process and were highly selective.The microchamber platform described here offers a cost-effective and sample-to-answer technology for on-site monitoring of multiple pathogens.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.

ABSTRACT
Miniaturized integrated DNA analysis systems have largely been based on a multi-chamber design with microfluidic control to process the sample sequentially from one module to another. This microchip design in connection with optics involved hinders the deployment of this technology for point-of-care applications. In this work, we demonstrate the implementation of sample preparation, DNA amplification, and electrochemical detection in a single silicon and glass-based microchamber and its application for the multiplexed detection of Escherichia coli and Bacillus subtilis cells. The microdevice has a thin-film heater and temperature sensor patterned on the silicon substrate. An array of indium tin oxide (ITO) electrodes was constructed within the microchamber as the transduction element. Oligonucleotide probes specific to the target amplicons are individually positioned at each ITO surface by electrochemical copolymerization of pyrrole and pyrrole-probe conjugate. These immobilized probes were stable to the thermal cycling process and were highly selective. The DNA-based identification of the two model pathogens involved a number of steps including a thermal lysis step, magnetic particle-based isolation of the target genomes, asymmetric PCR, and electrochemical sequence-specific detection using silver-enhanced gold nanoparticles. The microchamber platform described here offers a cost-effective and sample-to-answer technology for on-site monitoring of multiple pathogens.

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Related in: MedlinePlus

Photographs showing the silicon–glass microchip. (A) Upper left: top view of the silicon chip showing the fluidic holes along with thin-film platinum heater and temperature sensors; lower left: bottom view of the silicon chip showing the 8 μL reaction chamber and the through-hole for fluid introduction; right panel: glass chip with patterned indium tin oxide working electrodes. (B) The assembled silicon–glass microchip with pipet tips glued to the fluidic holes. (C) Electrical connection of the contact pins to the silicon–glass microchip housed in a plexiglass holder.
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fig1: Photographs showing the silicon–glass microchip. (A) Upper left: top view of the silicon chip showing the fluidic holes along with thin-film platinum heater and temperature sensors; lower left: bottom view of the silicon chip showing the 8 μL reaction chamber and the through-hole for fluid introduction; right panel: glass chip with patterned indium tin oxide working electrodes. (B) The assembled silicon–glass microchip with pipet tips glued to the fluidic holes. (C) Electrical connection of the contact pins to the silicon–glass microchip housed in a plexiglass holder.

Mentions: The silicon chip (thickness of 400 μm) had two fluid injection holes (top side, diameter of 500 μm, depth of 100 μm) and a chamber (bottom side, length and width of 5 mm, depth of 325 μm) etched by the inductively coupled plasma/deep reactive ion etching (ICP/DRIE) process, see Figure 1A (left panels). Thin-film platinum (100 nm) was patterned on top of the silicon substrate as heater and temperature sensors (Figure 1A, upper left). The glass chip had platinum pseudo-reference and counter electrodes (thickness of 100 nm) as well as four working electrodes made of indium tin oxide (ITO) (thickness of 100 nm), see right panel of Figure 1A. Ultra-violet curing optical cement (Type UV-69, Summers Optical, Hatfield, PA, USA) was used to bond the silicon and glass chips, the curing procedure was in accordance with the manufacturer's instruction. Pipet tips were glued to the fluid injection holes with epoxy (Figure 1B).


A DNA biochip for on-the-spot multiplexed pathogen identification.

Yeung SW, Lee TM, Cai H, Hsing IM - Nucleic Acids Res. (2006)

Photographs showing the silicon–glass microchip. (A) Upper left: top view of the silicon chip showing the fluidic holes along with thin-film platinum heater and temperature sensors; lower left: bottom view of the silicon chip showing the 8 μL reaction chamber and the through-hole for fluid introduction; right panel: glass chip with patterned indium tin oxide working electrodes. (B) The assembled silicon–glass microchip with pipet tips glued to the fluidic holes. (C) Electrical connection of the contact pins to the silicon–glass microchip housed in a plexiglass holder.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636451&req=5

fig1: Photographs showing the silicon–glass microchip. (A) Upper left: top view of the silicon chip showing the fluidic holes along with thin-film platinum heater and temperature sensors; lower left: bottom view of the silicon chip showing the 8 μL reaction chamber and the through-hole for fluid introduction; right panel: glass chip with patterned indium tin oxide working electrodes. (B) The assembled silicon–glass microchip with pipet tips glued to the fluidic holes. (C) Electrical connection of the contact pins to the silicon–glass microchip housed in a plexiglass holder.
Mentions: The silicon chip (thickness of 400 μm) had two fluid injection holes (top side, diameter of 500 μm, depth of 100 μm) and a chamber (bottom side, length and width of 5 mm, depth of 325 μm) etched by the inductively coupled plasma/deep reactive ion etching (ICP/DRIE) process, see Figure 1A (left panels). Thin-film platinum (100 nm) was patterned on top of the silicon substrate as heater and temperature sensors (Figure 1A, upper left). The glass chip had platinum pseudo-reference and counter electrodes (thickness of 100 nm) as well as four working electrodes made of indium tin oxide (ITO) (thickness of 100 nm), see right panel of Figure 1A. Ultra-violet curing optical cement (Type UV-69, Summers Optical, Hatfield, PA, USA) was used to bond the silicon and glass chips, the curing procedure was in accordance with the manufacturer's instruction. Pipet tips were glued to the fluid injection holes with epoxy (Figure 1B).

Bottom Line: Oligonucleotide probes specific to the target amplicons are individually positioned at each ITO surface by electrochemical copolymerization of pyrrole and pyrrole-probe conjugate.These immobilized probes were stable to the thermal cycling process and were highly selective.The microchamber platform described here offers a cost-effective and sample-to-answer technology for on-site monitoring of multiple pathogens.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.

ABSTRACT
Miniaturized integrated DNA analysis systems have largely been based on a multi-chamber design with microfluidic control to process the sample sequentially from one module to another. This microchip design in connection with optics involved hinders the deployment of this technology for point-of-care applications. In this work, we demonstrate the implementation of sample preparation, DNA amplification, and electrochemical detection in a single silicon and glass-based microchamber and its application for the multiplexed detection of Escherichia coli and Bacillus subtilis cells. The microdevice has a thin-film heater and temperature sensor patterned on the silicon substrate. An array of indium tin oxide (ITO) electrodes was constructed within the microchamber as the transduction element. Oligonucleotide probes specific to the target amplicons are individually positioned at each ITO surface by electrochemical copolymerization of pyrrole and pyrrole-probe conjugate. These immobilized probes were stable to the thermal cycling process and were highly selective. The DNA-based identification of the two model pathogens involved a number of steps including a thermal lysis step, magnetic particle-based isolation of the target genomes, asymmetric PCR, and electrochemical sequence-specific detection using silver-enhanced gold nanoparticles. The microchamber platform described here offers a cost-effective and sample-to-answer technology for on-site monitoring of multiple pathogens.

Show MeSH
Related in: MedlinePlus