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Microfluidic platform for isolating nucleic acid targets using sequence specific hybridization
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Image of FIG. 1.

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FIG. 1.

Microchip technique. (a) Illustration of magnetic bead capture complex of target ssDNA hybridized with BP onbead surface. (b) Illustration of thermodynamically simulated hybridization structures of target ssDNA and BP at hybridization condition. BP is highlighted in cyan shadow. (c) Illustration of target ssDNA separation by capture complex movement from reservoir W1 to W2 through a microchannel. (d) Design and photograph of a microfluidic chip. Reservoirs and channels are filled with dyes to enhance contrast. Channels are 3 cm long and 1 cm apart. Channels have tapered openings of 400 m wide at W1 and end with 50 m wide opening at W2. Depth of the channels is 120 m.

Image of FIG. 2.

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FIG. 2.

Bead exposed with BP. (a) Fluorescence measurements in reservoir W1 before and after beads were removed. (b)Concentration of BP adsorbed by the bead shown as a saturation curve. Y axis: number of BP per unit area (m) on bead surface.

Image of FIG. 3.

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FIG. 3.

Number of target ssDNA on bead with specific hybridization via BP (experimental data in red, model simulation in green) and non-specific adsorption to BP-2 (experimental data in magenta, model simulation in blue). Concentration of target ssDNA bond to bead is shown as number of target ssDNA per unit area (m) on bead surface.

Image of FIG. 4.

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FIG. 4.

NASBA-amplified detection of isolated target ssDNA with mixture of non-target ssDNA probe shown in real-time fluorescence as a function of time. Standard deviation is within 5% of results (error bars not shown). Note that 50% “contamination” of non-target ssDNA in sample solution made no difference in signal values compared to that of pure target ssDNA in detection.


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Table I.

Oligonucleotide sequences used in this study.


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The separation of target nucleic acid sequences from biological samples has emerged as a significant process in today's diagnostics and detection strategies. In addition to the possible clinical applications, the fundamental understanding of target and sequence specific hybridization on surface modified magnetic beads is of high value. In this paper, we describe a novel microfluidic platform that utilizes a mobile magnetic field in static microfluidic channels, where single stranded DNA (ssDNA) molecules are isolated via nucleic acid hybridization. We first established efficient isolation of biotinylated capture probe (BP) using streptavidin-coated magnetic beads. Subsequently, we investigated the hybridization of target ssDNA with BP bound to beads and explained these hybridization kinetics using a dual-species kinetic model. The number of hybridized target ssDNA molecules was determined to be about 6.5 times less than that of BP on the bead surface, due to steric hindrance effects. The hybridization of target ssDNA with non-complementary BP bound to bead was also examined, and non-specific hybridization was found to be insignificant. Finally, we demonstrated highly efficient capture and isolation of target ssDNA in the presence of non-target ssDNA, where as low as 1% target ssDNA can be detected from mixture. The microfluidic method described in this paper is significantly relevant and is broadly applicable, especially towards point-of-care biological diagnostic platforms that require binding and separation of known target biomolecules, such as RNA, ssDNA, or protein.


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Scitation: Microfluidic platform for isolating nucleic acid targets using sequence specific hybridization