الفهرس 
MicrofluidicsOnly 14 pages are availabe for public view
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Abstract
The use of microfluidics, portable devices in molecular diagnostics can offer several distinct advantages such as short assay times, low reagent consumption, as well as the potential to integrate multiple sample processing steps. Reproducibility and reliability for testing such microfluidics systems often depends on the ability to extract nucleic acids rapidly and efficiently. Isolation of nucleic acids such as DNA from biological samples is an essential step for genetic analysis.
To this end, DNA extraction method using a microfluidic silicon chip is proposed. The extraction by two methods was presented firstly a solid-phase DNA extraction and secondly sequence-specific extraction. For solid-phase DNA extraction, traditional chemical extraction techniques entail the use of centrifugation and organic solvents to purify DNA, making microfluidic integration difficult if not impossible. Newer filter based methods partially solve this problem. Filters consisting of well-ordered 5 to 10 μm silicon micro-pillars were fabricated and coated with chitosan.
As a comparison, and to optimize the DNA binding and elution, chitosan coated silica beads were initially used. Using such beads, up to 96% of bacteriophage lambda DNA could be trapped at pH 5 and about 25% of initial DNA could be eluted in a high salt buffer at pH 9. This protocol was successfully transferred to a continuous flow using the micro-pillar filter chips. Depending on the inter pillar distances, up to 95% capturing and up to ~30% elution was observed. To better understand the mode of DNA interaction with the chitosan coated filters, glutaraldehyde was used to crosslink all free amino groups present on the chitosan. Using this strategy, the capture efficiency significantly dropped to only around 20%, confirming the importance of the amino groups for efficient DNA binding. Based on the idea of binding of aldehyde group to amino group of chitosan-coated micropillars filter, a sequence-specific nucleic acid extraction on chitosan coated-silicon micropillar filters was presented.This method depends on DNA hybridization onto silicon-based micropillar coated with chitosan by the covalent coupling of aldehyde-functionalized DNA probes, which is a single strand DNA, recognized of complementary DNA target and formed a stable structure is the basis of highly specific bio recognizing devices. Chitosan-coated micropillar filters was coupled with 80.9% of aldehyde-functionalized DNA probe. About 70% of target DNA binds specifically to aldehyde probes of which 80% denatured from trapped DNA in NaOH buffer. About 17% of target DNA binds non-specifically to aldehyde probes of which 99% denatured from trapped DNA in NaOH buffer. To understand the mechanism of above interaction computational study is further conducted. Molecular modeling and Quantitative Structure Activity Relationship (QSAR) calculations were utilized at PM3 level in order to evaluate the interaction of aldehyde ssDNA on chitosan-functionalized silicon substrate and biological activity of the proposed compounds. Calculations were utilized at MM3 level in order to evaluate the interaction of target DNA on DNA probe on chitosan-functionalized silicon substrate through hydrogen bonding and biological activity of the proposed compounds. Calculated data indicate that, the more stable and reactive structures are DNA probe coupled on the chitosan-functionalized through hydrogen bond with SiO2 substrate through OH and NH of chitosan. While, the more reactive and rapidly forming hybridization with target DNA is the one that makes hydrogen bond with SiO2 through OH of chitosan. Finally the model molecules showed a correlation with the experimental data of DNA hybridization on chitosan-coated silicon micro-pillars.