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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 calibration plot of the sample-to-background ratio against the logarithmic number of E.coli cells in the sample. In our four working electrode design, the silver stripping time to reach a potential of +0.65 V (versus Pt pseudo-reference electrode) for the E.coli detection capture probe-modified electrodes was taken as the sample signal whereas that for the B.subtilis detection capture probe-modified electrodes was taken as the background signal. Note that the number of cells stated was the amount being introduced into the microchamber.
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fig5: A calibration plot of the sample-to-background ratio against the logarithmic number of E.coli cells in the sample. In our four working electrode design, the silver stripping time to reach a potential of +0.65 V (versus Pt pseudo-reference electrode) for the E.coli detection capture probe-modified electrodes was taken as the sample signal whereas that for the B.subtilis detection capture probe-modified electrodes was taken as the background signal. Note that the number of cells stated was the amount being introduced into the microchamber.

Mentions: The ability of this single microchamber to detect specific cell type using the above protocol is demonstrated by running a series of experiments with E.coli cells of different concentrations, taking the signal from the B.subtilis detection capture probe-modified electrode as the background. Figure 5 gives a semi-log plot of the sample to background ratio against the number of cells in the sample. A linear relationship is obtained in the concentration range investigated (102–105 cells/sample). This result confirms the successful isolation of the genome with the magnetic particle-based approach, compatibility of the magnetic particle with the PCR, thermal stability of the immobilized detection capture probe through the temperature cycling process, as well as negligible nonspecific adsorption on the electrode surface.


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

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

A calibration plot of the sample-to-background ratio against the logarithmic number of E.coli cells in the sample. In our four working electrode design, the silver stripping time to reach a potential of +0.65 V (versus Pt pseudo-reference electrode) for the E.coli detection capture probe-modified electrodes was taken as the sample signal whereas that for the B.subtilis detection capture probe-modified electrodes was taken as the background signal. Note that the number of cells stated was the amount being introduced into the microchamber.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: A calibration plot of the sample-to-background ratio against the logarithmic number of E.coli cells in the sample. In our four working electrode design, the silver stripping time to reach a potential of +0.65 V (versus Pt pseudo-reference electrode) for the E.coli detection capture probe-modified electrodes was taken as the sample signal whereas that for the B.subtilis detection capture probe-modified electrodes was taken as the background signal. Note that the number of cells stated was the amount being introduced into the microchamber.
Mentions: The ability of this single microchamber to detect specific cell type using the above protocol is demonstrated by running a series of experiments with E.coli cells of different concentrations, taking the signal from the B.subtilis detection capture probe-modified electrode as the background. Figure 5 gives a semi-log plot of the sample to background ratio against the number of cells in the sample. A linear relationship is obtained in the concentration range investigated (102–105 cells/sample). This result confirms the successful isolation of the genome with the magnetic particle-based approach, compatibility of the magnetic particle with the PCR, thermal stability of the immobilized detection capture probe through the temperature cycling process, as well as negligible nonspecific adsorption on the electrode surface.

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