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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

A schematic representation of the assay protocol in the silicon−glass microchamber. The three main steps were (A) sample preparation: thermal cell lysis and magnetic particle-based isolation of specific genomic DNAs; (B) target DNA amplification: generation of single-stranded rich amplicons by asymmetric PCR; (C) product detection: gold nanoparticle labeling, electrocatalytic silver deposition, and electrochemical silver dissolution.
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fig4: A schematic representation of the assay protocol in the silicon−glass microchamber. The three main steps were (A) sample preparation: thermal cell lysis and magnetic particle-based isolation of specific genomic DNAs; (B) target DNA amplification: generation of single-stranded rich amplicons by asymmetric PCR; (C) product detection: gold nanoparticle labeling, electrocatalytic silver deposition, and electrochemical silver dissolution.

Mentions: The assay procedure used in this work is schematically represented in Figure 4. It involves three main steps: sample preparation, target DNA amplification, and product detection, all performed within the same microchamber. Intact cells are first broken down by applying a high temperature (90°C, controlled by the on-chip heater and temperature sensor) to free the genomic DNA. To remove all the interfering substances (e.g. cell debris and protein) that may affect the subsequent DNA amplification process, magnetic particles are used to isolate the specific genomes. Biotinylated genome capture probes for the two model species are mixed with the intact cells before injecting into the microchamber. When the temperature is lowered to 50°C after the thermal lysis step, these probes hybridize to their complementary target genomes. These probe−genome hybrids are then isolated by the addition of the avidin-coated magnetic particles, followed by thorough washing. It is worth noting that the magnetic particles are pretreated with a small amount of the genome capture probes to minimize nonspecific adsorption of the interfering substances and other genomic DNAs. Subsequently, with the genomes captured on the magnetic particles serving as the template, asymmetric PCR is conducted to generate single-stranded rich target amplicons. After the amplification step, these amplicons hybridize to their corresponding detection electrodes. Next, the hybridized amplicons are labeled with gold nanoparticles via biotin–avidin interaction. Finally, silver metal is electrocatalytically deposited onto the gold nanoparticles and the amount is determined by the electrochemical oxidative dissolution technique (10,12). The detailed procedure for the sample preparation, target DNA amplification, and product detection steps is given in the Materials and Methods section.


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

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

A schematic representation of the assay protocol in the silicon−glass microchamber. The three main steps were (A) sample preparation: thermal cell lysis and magnetic particle-based isolation of specific genomic DNAs; (B) target DNA amplification: generation of single-stranded rich amplicons by asymmetric PCR; (C) product detection: gold nanoparticle labeling, electrocatalytic silver deposition, and electrochemical silver dissolution.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: A schematic representation of the assay protocol in the silicon−glass microchamber. The three main steps were (A) sample preparation: thermal cell lysis and magnetic particle-based isolation of specific genomic DNAs; (B) target DNA amplification: generation of single-stranded rich amplicons by asymmetric PCR; (C) product detection: gold nanoparticle labeling, electrocatalytic silver deposition, and electrochemical silver dissolution.
Mentions: The assay procedure used in this work is schematically represented in Figure 4. It involves three main steps: sample preparation, target DNA amplification, and product detection, all performed within the same microchamber. Intact cells are first broken down by applying a high temperature (90°C, controlled by the on-chip heater and temperature sensor) to free the genomic DNA. To remove all the interfering substances (e.g. cell debris and protein) that may affect the subsequent DNA amplification process, magnetic particles are used to isolate the specific genomes. Biotinylated genome capture probes for the two model species are mixed with the intact cells before injecting into the microchamber. When the temperature is lowered to 50°C after the thermal lysis step, these probes hybridize to their complementary target genomes. These probe−genome hybrids are then isolated by the addition of the avidin-coated magnetic particles, followed by thorough washing. It is worth noting that the magnetic particles are pretreated with a small amount of the genome capture probes to minimize nonspecific adsorption of the interfering substances and other genomic DNAs. Subsequently, with the genomes captured on the magnetic particles serving as the template, asymmetric PCR is conducted to generate single-stranded rich target amplicons. After the amplification step, these amplicons hybridize to their corresponding detection electrodes. Next, the hybridized amplicons are labeled with gold nanoparticles via biotin–avidin interaction. Finally, silver metal is electrocatalytically deposited onto the gold nanoparticles and the amount is determined by the electrochemical oxidative dissolution technique (10,12). The detailed procedure for the sample preparation, target DNA amplification, and product detection steps is given in the Materials and Methods section.

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