The long-term goal of this NIH research project is to demonstrate a novel nanotechnology concept that we developed at Oak Ridge National Laboratory for rapid nanoscale reading of nucleic acid sequences on an individual molecule. According to this concept, it is possible to obtain genetic sequence information by probing through a DNA molecule base by base at a nanometer scale - as if looking through a strip of movie film. The proposed nanotechnology has the potential capability of performing DNA sequencing at a speed at least 2800 times faster than that of the current technology: that is, a sequencing job that would take 2000 years to complete using the current machine could be accomplished within 1 year via this nanotechnology. This enhanced performance is made possible by a series of innovations, including novel applications of a fine-tuned nanometer gap for passage of a single DNA molecule, thin-layer microfluidics for sample loading and delivery, programmable electric fields for precise control of DNA movement, and detection of DNA nucleotide bases by nanoelectrode-gated tunneling conductance measurements. One of the most crucial components is the nanometer nucleotide detection gate, which comprises two sharp tips of nanoelectrodes pointing toward each other on a nonconductive (e.g., SiO2) plate. At the R21 pilot phase (the first two years) of this experimental project, we will fabricate this detection gate using electron-beam lithography and our patented programmable pulsed precision electrolytic nanofabrication technique. We will also perform proof-of-principle demonstration for detection of nucleotide bases such as poly [A] or poly [C] in a 2- to 5-nm electrode gap by tunneling conductance spectroscopic measurements across the nanoelectrode gate. When we achieve these R21 milestones (fabrication of nanoelectrode detection gate and proof-of-principle demonstration for detection of nucleotide bases) to the satisfaction of NIH, this project may then be determined to move onto its development phase (R33) to fully develop and demonstrate this novel nanotechnology for rapid DNA sequencing by nanoscale direct reading on single DNA molecules. This project is expected to deliver a prototype of the envisioned rapid sequencing nanotechnology near the end of its R33 phase. This (R21/R33) R&D project proposal is in response to NIH RFA-HG-04-003 program announcement for Revolutionary Genome Sequencing Technologies. ? ?