While the cost of DNA sequencing has dropped significantly over the last few years due to the evolution of next-generation sequencing instruments, there still exists the need to produce new technologies that can significantly reduce sequencing cost and time and improve the level of automation to realize the ability of transitioning DNA sequencing into currently inaccessible areas, such as the clinic for in vitro diagnostics. In fact, reaching the goals mandated by the $1,000 Genome Project will provide the ability to use DNA sequencing as a de facto standard for looking at any sequence variation over the entire genome. The long term goal of this project is to generate a novel DNA sequencing platform that can substantially reduce the cost, labor and time associated with acquiring DNA sequencing information using a fully automated platform. The strategy uses nano-scale sensors that read the identity of mononucleotide bases from their characteristic flight- time through a 2-dimensional (2D) nanochannel (<10 nm in width and depth;>5 ?m in length) fabricated in a thermoplastic, such as Plexiglas, via low-cost nanoimprint lithography and other replication-based techniques. The mononucleotide bases are generated from an intact DNA fragment (~50,000 bp) using a processive exonuclease, which is covalently anchored to a support contained within a bioreactor that feeds the mononucleotides into the 2D nanochannel. The identity of the mononucleotide is deduced from a molecular- dependent flight-time through the 2D nanochannel. The major focus of this R21 application is to develop a transduction modality that can measure the flight-time of mononucleotides through a 2D polymer nanochannel without requiring a reporter molecule covalently attached to the mononucleotide. The transducer to be investigated consists of 2 pairs of nanoelectrodes poised at each end of the 2D nanochannel with the signal resulting from perturbations in the conductivity induced by the mononucleotide. The sensing platform is produced from nanowires built using templating methods from anodized aluminum oxide materials and then, electrochemically thinned to the desired diameter (~10 nm). The wires are strategically placed on a nanofluidic chip using chemical patterns made via nanoimprint lithography with the required gap (<10 nm) generated via mechanical or chemical steps. The nanosensor chips are produced on a plastic module that can be integrated via novel interconnect technologies to other DNA processing modules to provide complete automation of the DNA sample processing pipeline. The envisioned DNA sequencing platform will produce ~1 x 106 nucleotide base reads s-1 when configured in an arrayed format, process an entire sample in a fully automated fashion with the cost of the modular fluidic system <$200. The low-cost of the fluidic system results not only from the use of replication technologies to produce the fluidic network spanning over multiple size scales, but also the simple and highly parallel strategies used to produce the nano-scale components required for this chip.
A novel single-molecule DNA sequencing system is envisioned that utilizes a modular 3D approach to process input DNA with each module spanning several size domains (mm ? nm). One module is a nanosensor chip, which is comprised of 2D nanochannels used to identify individual mononucleotides through their molecular- dependent flight-time through the nanochannel. The flight-time is transduced using single-molecule conductivity measurements, which is measured using nano-electrodes poised at the input and output ends of the nanochannel. In this R21 application, the feasibility of measuring the conductivity response of single mononucleotides will be demonstrated.
|Weerakoon-Ratnayake, Kumuditha M; O'Neil, Colleen E; Uba, Franklin I et al. (2017) Thermoplastic nanofluidic devices for biomedical applications. Lab Chip 17:362-381|
|Weerakoon-Ratnayake, Kumuditha M; Uba, Franklin I; Oliver-Calixte, Nyoté J et al. (2016) Electrophoretic Separation of Single Particles Using Nanoscale Thermoplastic Columns. Anal Chem 88:3569-77|
|ONeil, Colleen E; Jackson, Joshua M; Shim, Sang-Hee et al. (2016) Interrogating Surface Functional Group Heterogeneity of Activated Thermoplastics Using Super-Resolution Fluorescence Microscopy. Anal Chem 88:3686-96|
|Uba, Franklin I; Hu, Bo; Weerakoon-Ratnayake, Kumuditha et al. (2015) High process yield rates of thermoplastic nanofluidic devices using a hybrid thermal assembly technique. Lab Chip 15:1038-49|
|Uba, Franklin I; Pullagurla, Swathi R; Sirasunthorn, Nichanun et al. (2015) Surface charge, electroosmotic flow and DNA extension in chemically modified thermoplastic nanoslits and nanochannels. Analyst 140:113-26|
|Oliver-Calixte, Nyoté J; Uba, Franklin I; Battle, Katrina N et al. (2014) Immobilization of lambda exonuclease onto polymer micropillar arrays for the solid-phase digestion of dsDNAs. Anal Chem 86:4447-54|
|Chantiwas, Rattikan; Park, Sunggook; Soper, Steven A et al. (2011) Flexible fabrication and applications of polymer nanochannels and nanoslits. Chem Soc Rev 40:3677-702|