Dynamic DNA hybridization is presented as an approach to perform gene expression analysis. The method is advantageous because of its dynamic supplies of both DNA samples and probes. The approach was demonstrated on a microfluidic platform by incorporating paramagnetic beads as a transportable solid support. A glass chip was fabricated to allow simultaneous interrogation of eight DNA target samples by DNA probes. DNA targets were immobilized on beads via streptavidin-biotin conjugation or base pairing between oligonucleotide residues. The DNA/bead complex was introduced into the device in which hybridization took place with a complementary probe. The hybridized probe was then removed by heat denaturation to allow the DNA sample to be interrogated again by another probe with a different sequence of interest. A pneumatic pumping apparatus was constructed to transport DNA probes and other reagents into the microfluidic device while hydrostatic pumping was used for the introduction of paramagnetic beads with samples. After investigating three types of paramagnetic beads, we found Dynabeads Oligo(dT)25 best suited this application. Targets on the beads could be sequentially interrogated by probes for 12 times, and the hybridization signal was maintained within experimental variation. Demonstration of specific hybridization reactions in an array format was achieved using four synthesized DNA targets in duplicate and five probes in sequence, indicating the potential application of this approach to gene expression analysis.

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A microfluidic assay was developed for screening botulinum neurotoxin serotype A (BoNT-A) by using a fluorescent resonance energy transfer (FRET) assay. Molded silicone microdevices with integral valves, pumps, and reagent reservoirs were designed and fabricated. Electrical and pneumatic control hardware were constructed, and software was written to automate the assay protocol and data acquisition. Detection was accomplished by fluorescence microscopy. The system was validated with a peptide inhibitor, running 2 parallel assays, as a feasibility demonstration. The small footprint of each bioreactor cell (0.5 cm2) and scalable fluidic architecture enabled many parallel assays on a single chip. The chip is programmable to run a dilution series in each lane, generating concentration-response data for multiple inhibitors. The assay results showed good agreement with the corresponding experiments done at a macroscale level. Although the system has been developed for BoNT-A screening, a wide variety of assays can be performed on the microfluidic chip with little or no modification.

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