Single molecule DNA/RNA sequencing technology based on a parallel Raman scattering readout in a coupled nanochannel/nanopore system

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Single molecule DNA/RNA transport and Raman sequencing technology based on parallel Raman scattering readout in a coupled nanochannel/nanopore system. Abstract Armonica Technologies, Inc. is proposing to develop a novel, high-throughput, label-free, highly accurate, long-read DNA sequencing platform based on inexpensive nanoscale patterning and self- assembly. The platform consists of nanochannels (cross section dimensions of ~ 100nm); tortuous (convoluted 3D) nanopores formed by self-assembly of colloidal nanoparticles; nanoparticle barriers placed across the nanochannels; and a metal-insulator-metal (MIM) field enhancement structure atop the nanochannel roof. In operation, single- stranded- or double-stranded-DNA is partially stretched into a linear configuration in the nanochannels, is blocked at barriers incorporated into the channels and forced (by electric field) to translocate through the tortuous nanopores in the roof. The MIM structure on the roof locally enhances the electromagnetic fields of applied laser sources allowing surface enhanced coherent anti-Stokes Raman scattering (SECARS) detection of individual bases as they pass through the electromagnetic hot spots, thus providing single base sensitivity and spatial localization. The distinct Raman spectra of the individual bases and epigenetic variants allow label-free sequencing. Optical detection allows massively parallel operation since the only requirement is separation of the pores by more than an optical wavelength, easily accomplished in the fabrication. An important feature of the platform is that the porous roofs allow introduction of oligonucleotides, small proteins, and DNA- binding/DNA-processing enzymes, permitting optional manipulation and modification of the DNA in the nanochannels. The goals of this Phase II project are: to develop a protocol for electrophoretic control of the ssDNA translocation; to engineer the structure to ensurethat the ssDNA passes the MIM hot spot; to sequence a short section of ssDNA and evaluate error rates; and to develop parallel optical readout, AI analysis of the data and appropriate storage protocol.