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We report the surface modification of thermoplastic nanochannels and the evaluation of the surface charge density, zeta potential and electroosmotic flow (EOF).

The process of immobilizing enzymes onto solid supports for bioreactions has some compelling advantages compared to their solution-based counterpart including the facile separation of enzyme from products, elimination of enzyme autodigestion, and increased enzyme stability and activity. We report the immobilization of λ-exonuclease onto poly(methylmethacrylate) (PMMA) micropillars populated within a microfluidic device for the on-chip digestion of double-stranded DNA. Enzyme immobilization was successfully accomplished using 3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling to carboxylic acid functionalized PMMA micropillars. Our results suggest that the efficiency for the catalysis of dsDNA digestion using λ-exonuclease, including its processivity and reaction rate, were higher when the enzyme was attached to a solid support compared to the free solution digestion. We obtained a clipping rate of 1.0 × 103 nucleotides s–1 for the digestion of λ-DNA (48.5 kbp) by λ-exonuclease. The kinetic behavior of the solid-phase reactor could be described by a fractal Michaelis–Menten model with a catalytic efficiency nearly 17% better than the homogeneous solution-phase reaction. The results from this work will have important ramifications in new single-molecule DNA sequencing strategies that employ free mononucleotide identification.

Fluidic devices that possess structures less than 150 nm in one or two dimensions are generating great interest due to the unique properties afforded by this size domain not accessible at the microscale. As molecules travel through nanochannels, they undergo hydrophobic and van der Waals interactions with the channel walls at a degree that depends on the size of the channel, the surface chemistry of the wall and the debye length (governed by the ionic strength of the electrolyte solution). In this work, we report the fabrication of nanometer sized structures (nanoslits, nanochannels and nanoelectrodes) in thermoplastic and fused silica substrates for the analysis of dsNA. In the case of thermoplastics, mixed-scale micro- and nanofluidic networks were fabricated using a simple, high resolution, single-step thermal embossing process and the fluidic structures were enclosed via low temperature fusion bonding to a cover plate. Nanochannels were chemically modified and the associated electrokinetic parameters - surface charge density, zeta potential and electroosmotic flow - were evaluated. In the fused silica substrate, we developed an integrated nanosensing device comprising of a single nanochannel and two pairs of transverse electron-conducting (~50 × 50 nm) nanoelectrodes separated by a nanometer gap (nanogap) and poised at the input and output ends of the nanochannel. This device serves a foundation for a novel technique we developing for the sequencing of DNA molecule by measuring the transit time of the monomer units entering and exiting a nanochannel (5 to 50 nanochannels) after being clipped from a single polymer digested with an enzyme. Further experiments on single molecule electrophoresis will provide information on possible routes that can be adopted to engineer proper nanochannel wall chemistry for the enhancement or reduction of solute/wall interactions.