Characterization of Multilayer Microfluidics for Biomedical Applications

Abstract

In this study the design and performance of a microfluidic silicon chip capable of continuous flow capture and elution of DNA was presented.The micro-pillars had a high-aspect ratio (HxD: 300 µm x ~25 µm) resulting in a significantly increased surface to volume ratio (e.g. 6.6 cm2 total area for filters with an IPD of 10 µm). As an alternatively, capillaries packed with silica microparticles have been described to provide massive surface to volume ratios, but their stacking typically results in very high pressure drops making it less attractive for portable chip tests. In this work, we combined the high surface area of the micro-pillars with a chitosan coating. 95% of the DNA was effectively captured by the chitosan-coated beads. Based on the protocol used for DNA binding and elution on chitosan coated beads, similar tests were performed on chips with chitosan-coated micro-pillars. The DNA trapped was 96% and elution efficiency was 25 % using Tris buffer at pH 9. The effect of lowering the amount of amino groups was tested by cross linking of the amino-groups using different concentrations of glutaraldehyde. The ratios of DNA eluted/DNA trapped increased with increasing glutaraldehyde /chitosan ratios. In conclusion, the results shown in this work provide the use of a miniaturized micro-pillar filter for future use in a portable device with integrated sample preparation. The high aspect ratio of the filters allow to generate rapid purified DNA using low pressures and limited reagent consumption. The DNA hybridization event in which the probe, which is usually a single-stranded DNA covalently immobilized on a functionalized surface, recognizes the complementary target and forms a stable duplex structure is the basis of highly specific bio recognizing devices. A nucleic acid hybridization assay was assembled onto silicon-based micropillar coated with chitosan as a scaffold for the covalent coupling of aldehyde-functionalized DNA probes. Chitosan-coated micropillar filters was coupled with 80.9% of aldehyde-functionalized DNA probe.About 70% of target DNA binds specifically to aldehyde-functionalized DNA probes of which 80% denatured from trapped DNA in NaOH buffer. About17% of target DNA binds non-specifically to aldehyde-functionalized poly-T probes of which 99% denatured from trapped DNA in NaOH buffer. The future work will contribute to the development of MPF devices for sequence-specific nucleic acid purification from whole blood. Molecular modeling calculation were used to study the interaction of hybridization of a small four base pair double strand DNA starting from single strands on chitosan functionalized silicon dioxide substrate. One strand was coupled to a chitosan functionalized silicon dioxide substrate calculated at PM3 and the other was hybridized on this structure calculated at MM3. The more stable and reactive structures calculated at PM3, are DNA probe coupled on the chitosan-functionalized through hydrogen bond with SiO2substrate through OH and NH of chitosan. According to MM3 calculation, the more reactive and rapidly forming hybridization with target DNA is the one that makes hydrogen bond with SiO2 through OH of chitosan.