Microfluidic platform for isolating nucleic acid targets using sequence specific hybridization

Abstract

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.

A Simple Microfluidic Assay for the Detection of Ligation Product

“…We experienced a higher bead transfer efficiency and lower analyte loss compared with off-chip techniques. Our previous publications [9][10][11] demonstrate that the chip design utilized in this assay resulted in nearly 100 % bead transfer efficiency. In addition, microfluidic assay results in a lower analyte handling volume.…”

“…Streptavidin-coated magnetic beads (Dynabead Ò M-280, Invitrogen TM ) were first washed and saturated with capture The site of mismatch is highlighted in bold probes by mixing in hybridization buffer and incubated for 30 min at room temperature, as previously reported [10,11]. Excessive capture probes were washed out and 1 lg of capture probe-saturated beads were added into the ligation mixture ( Table 2).…”

“…The microchips were fabricated using soft lithography, as previously reported [10,11]. Polydimethylsiloxane (PDMS) chips were generated over a master mold (fabricated SU-8 microchannel structure on a silicon wafer) and bonded to a Gold Seal Ò glass slide.…”

An automated microfluidic system for single-stranded DNA preparation and magnetic bead-based microarray analysis

“…In the late 1990s, electric fields were first reported to concentrate negatively charged DNA strands to probes on electrodes and the hybridization process was thus accelerated. 14 Since then, a number of mechanisms, such as hydrodynamic flow, 10,15 acoustic wave, 16 electrokinetic, 17 and magnetic deliveries, 18,19 have been developed to transform the static microarray into a microfluidic format in order to overcome the diffusion limit of the hybridization. Lee et al developed a recirculating microfluidic device that repeatedly flowed samples to a probe array using a peristaltic pump to reduce the hybridization time from 6 to 2 h. 20 Recently, Karsenty et al employed an isotachophoretic method to focus targets to arrays in a straight channel, demonstrating two orders of magnitude improvement in limit of detection.…”

Integration of programmable microfluidics and on-chip fluorescence detection for biosensing applications

“…Microfluidic assays include nonspecific silica based nucleic acid adsorption, 16 antibody mediated nucleic acid isolation, 17 and bead-based sequence specific hybridization extraction. 18 Optimization of assays and microfluidic/optofluidic functionality simultaneously is possible through hybrid integration in which a sample handling and a sensing layer are designed independently in terms of materials and functionality. Recently, hybrid systems combining PDMS (polydimethylsiloxane)-based microfluidic layers with a liquid-core waveguide optical detection layer have been reported.…”