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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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(A–D) A schematic diagram showing the electrode-by-electrode capture probe immobilization via electrochemical copolymerization of pyrrole and pyrrole−oligonucleotide. The polymerization solution contained 60 mM pyrrole, 20 μM pyrrole−probe, and 0.1 M LiClO4 and the electrode was subjected to a cyclic voltammetric scan between −0.5 and +0.65 V (versus Pt pseudo-reference electrode) at a scan rate of 50 mV/s for three times.
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fig2: (A–D) A schematic diagram showing the electrode-by-electrode capture probe immobilization via electrochemical copolymerization of pyrrole and pyrrole−oligonucleotide. The polymerization solution contained 60 mM pyrrole, 20 μM pyrrole−probe, and 0.1 M LiClO4 and the electrode was subjected to a cyclic voltammetric scan between −0.5 and +0.65 V (versus Pt pseudo-reference electrode) at a scan rate of 50 mV/s for three times.

Mentions: The single microchamber design poses particular challenges to the electrochemical platform used for the sequence-specific PCR amplicons detection. Addressability and compatibility are two important considerations regarding immobilization chemistry for the oligonucleotide detection capture probes. For a multiplexed assay, it is necessary to individually modify the detection platform so that each individual electrode in an electrode array has a specific capture probe. When using either high temperature or ultra-violet glue to seal the microchamber, it is recommended that immobilization should be carried out after the silicon−glass bonding process so as to prevent damage to the capture probes. In doing so, the more common chemical attachment (spotting) method cannot be used because all the active electrode surfaces are embedded within the same microchamber and they would receive identical modifications. One simple way to achieve site-specific probe immobilization onto individual electrode surfaces can be achieved by electrochemical copolymerization of pyrrole and pyrrole−oligonucleotide (11). Figure 2 illustrates the strategy to immobilize different capture probes onto each individual electrode. A solution of pyrrole and oligonucleotide 1 bearing a pyrrole group is introduced into the microchamber. When a cyclic voltammetric scan is applied to electrode 1, with other electrodes disconnected or grounded, oligonucleotide 1 is selectively deposited on this particular electrode. Then, the microchamber is washed with water to ensure there is no pyrrole−oligonucleotide 1 monomer is left. This procedure is repeated for the other electrodes with different pyrrole−oligonucleotide polymerization solutions. In our model system with two target analytes and four working electrodes, the capture probes specific to E.coli and B.subtilis are immobilized in duplicate. Before proceeding to the complete analytical protocol, the ability of these immobilized capture probes to recognize their complementary targets should be tested. Figure 3 shows the fluorescence images of the four functionalized electrodes (A and D: B.subtilis probe; B and C: E.coli probe) exposed to a sample containing a fluorescently-labeled sequence complementary to the E.coli probe. It is clear that electrodes B and C exhibit much higher fluorescence intensity than electrodes A and D, indicating the highly specific probe immobilization as well as hybridization offered by the electrochemical pyrrole-based attachment chemistry. Another criterion for the selection of immobilization method is the compatibility with other processes, in particular the PCR. Due to the fact that the detection electrodes are within the reaction chamber, the linkage between the immobilized capture probe and electrode surface must be strong enough to survive through the thermal cycling process (especially the high denaturation temperature). Moreover, the detector surface should interact only with the specific amplicon but not with other components employed in the assay protocol.


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

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

(A–D) A schematic diagram showing the electrode-by-electrode capture probe immobilization via electrochemical copolymerization of pyrrole and pyrrole−oligonucleotide. The polymerization solution contained 60 mM pyrrole, 20 μM pyrrole−probe, and 0.1 M LiClO4 and the electrode was subjected to a cyclic voltammetric scan between −0.5 and +0.65 V (versus Pt pseudo-reference electrode) at a scan rate of 50 mV/s for three times.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: (A–D) A schematic diagram showing the electrode-by-electrode capture probe immobilization via electrochemical copolymerization of pyrrole and pyrrole−oligonucleotide. The polymerization solution contained 60 mM pyrrole, 20 μM pyrrole−probe, and 0.1 M LiClO4 and the electrode was subjected to a cyclic voltammetric scan between −0.5 and +0.65 V (versus Pt pseudo-reference electrode) at a scan rate of 50 mV/s for three times.
Mentions: The single microchamber design poses particular challenges to the electrochemical platform used for the sequence-specific PCR amplicons detection. Addressability and compatibility are two important considerations regarding immobilization chemistry for the oligonucleotide detection capture probes. For a multiplexed assay, it is necessary to individually modify the detection platform so that each individual electrode in an electrode array has a specific capture probe. When using either high temperature or ultra-violet glue to seal the microchamber, it is recommended that immobilization should be carried out after the silicon−glass bonding process so as to prevent damage to the capture probes. In doing so, the more common chemical attachment (spotting) method cannot be used because all the active electrode surfaces are embedded within the same microchamber and they would receive identical modifications. One simple way to achieve site-specific probe immobilization onto individual electrode surfaces can be achieved by electrochemical copolymerization of pyrrole and pyrrole−oligonucleotide (11). Figure 2 illustrates the strategy to immobilize different capture probes onto each individual electrode. A solution of pyrrole and oligonucleotide 1 bearing a pyrrole group is introduced into the microchamber. When a cyclic voltammetric scan is applied to electrode 1, with other electrodes disconnected or grounded, oligonucleotide 1 is selectively deposited on this particular electrode. Then, the microchamber is washed with water to ensure there is no pyrrole−oligonucleotide 1 monomer is left. This procedure is repeated for the other electrodes with different pyrrole−oligonucleotide polymerization solutions. In our model system with two target analytes and four working electrodes, the capture probes specific to E.coli and B.subtilis are immobilized in duplicate. Before proceeding to the complete analytical protocol, the ability of these immobilized capture probes to recognize their complementary targets should be tested. Figure 3 shows the fluorescence images of the four functionalized electrodes (A and D: B.subtilis probe; B and C: E.coli probe) exposed to a sample containing a fluorescently-labeled sequence complementary to the E.coli probe. It is clear that electrodes B and C exhibit much higher fluorescence intensity than electrodes A and D, indicating the highly specific probe immobilization as well as hybridization offered by the electrochemical pyrrole-based attachment chemistry. Another criterion for the selection of immobilization method is the compatibility with other processes, in particular the PCR. Due to the fact that the detection electrodes are within the reaction chamber, the linkage between the immobilized capture probe and electrode surface must be strong enough to survive through the thermal cycling process (especially the high denaturation temperature). Moreover, the detector surface should interact only with the specific amplicon but not with other components employed in the assay protocol.

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