Selective DNA Sensing Elements Integrated into Microfluidic Channels
 

Rebecca A. Zangmeister, and Michael J. Tarlov

Process Measurements Division, Chemical Science and Technology Laboratory
National Institute of Standards and Technology, Gaithersburg, Maryland 20899

The maturation of microchip technology coupled with traditional bioassays has led to recent advances in the field of bioanalytical analysis. Microfluidic devices, one such platform, offer analytical advantages for samples of low concentration and small sample volume due to directed, efficient mass transport of analytes through microfluidic networks. Although significant advances have been made in DNA detection using array devices, there exists a need for a high throughput, diagnostic DNA detection system. One such strategy is to incorporate a selective DNA screening element into a microfluidic device that takes advantage of the aforementioned attributes.

We describe here a method for immobilizing single-stranded DNA (ss-DNA) probe molecules in polyacrylamide hydrogels within plastic microfluidic channels. The plugs are formed by photopolymerization of a solution containing 19:1 polyacrylamide/bisacrylamide and ss-DNA modified at the 5' end with an acrylic acid group. Short illumination times (5 min) are used, and spatial definition is achieved by the size and shape of the light source aperture. Fluorescent-tagged ss-DNA targets are driven through the hydrogel plugs using electrophoresis, and hybridize with complementary ss-DNA probes covalently bound in the hydrogel. The efficient hybridization of ss-DNA targets, coupled with the directed mass transfer associated with of the microchannel result in an effective selective detection element within a microfluidic channel.

A unique diagnostic assay has been developed that is based on the displacement of a sacrificial fluorescent-tagged indicator sequence by a target sequence of interest. The distinct advantage of such an assay is that the target DNA sequence does not have to be pre-labeled prior to analysis. Two plugs of a DNA/hydrogel copolymer are formed in series in a plastic microchannel (Figure 1). The first plug is loaded with a fluorescent-tagged 10 mer indicator sequence that is complementary to one half of the immobilized probe sequence. Labeled and unlabelled target oligomers of 20 base pairs are electrophoresed through the microchannel. The 20 mer, complementary to the immobilized probe sequence, displaces the 10 mer indicator sequence because of the differences in the calculated DG of formation for each hybrid pair (-133.7 kJ mol-1 vs. -47.8 kJ mol-1 respectively), and is detected in the second capture plug. A control, non-complementary DNA sequence passes through the 1st plug without displacement of the indicator 10 mer sequence. Results from single and dual color assays will be presented, along with future experimental designs.