Plastic-based Nanofluidic Devices for Single Molecule DNA/RNA Sequencing
Steve Soper,
Foundation Distinguished Professor; Director, Center of BioModular Multi-scale System for Precision Medicine, Adjunct Professor, Ulsan National Institute of Science & Technology,
The University of Kansas
We are generating a single-molecule, amplification-free DNA/RNA sequencing platform that can acquire sequencing information with high accuracy (>95%) at unprecedented throughputs (106 bases s-1). The technology employs high density arrays of nanochannels that read the identity of individual mononucleotides from their molecular-dependent electrophoretic mobilities through a 2-dimensional (2D) nanochannel (~50 nm in width and depth; >10 µm in length) fabricated in a thermoplastic via nanoimprint lithography (NIL) or injection compression molding. The single mononucleotides are generated from an intact DNA fragment using a highly processive exonuclease, which is covalently anchored to a plastic solid support contained within a bioreactor that sequentially feeds cleaved mononucleotides into the 2D nano-electrophoresis channel. The identity of each mononucleotide is deduced from its molecular-dependent electrophoretic mobility through the 2D nanochannel. The mobility is read in a label-free fashion by measuring current transients (i.e., resistive pulse sensing) induced by a single mononucleotide when it travels through a constriction with molecular dimensions (<10 nm in effective diameter) poised at the input/output ends of the electrophoresis channel.
In this presentation, our results in using nanoscale electrophoresis to deduce the identity of both the deoxynucleotides and ribonucleotides will be discussed, especially material and scaling effects on the performance of nano-electrophoresis. Also, different surface modification strategies of thermoplastics will be presented that alter the electroosmostic flow and its effects on separation performance. I will also discuss the surface immobilization of exonucleases onto solid-plastic supports using UV/O3 activation with EDC/NHS coupling chemistry. In particular, the effects of surface immobilization on enzyme kinetic rates, processivity, and stability will be discussed. Finally, the fabrication and operation of in-plane nanopore sensors to detect single molecules will be discussed.
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