Project Summary To improve DNA and RNA sequencing with respect to accuracy, robustness and speed, this NIH R21 project focuses on using a two-layer design with two parallel solid-state SiN-2D nanopores on low-noise all-glass chips, towards DNA sequencing and direct RNA sequencing. The basic concept behind nanopores involves using an applied voltage to drive single-stranded DNA molecules through a narrow nanopore, which separates chambers of electrolyte solution. This voltage also drives a flow of electrolyte ions through the pore, measured as an electric current. When molecules pass through the nanopore they modify the flow of ions, and structural information can be extracted by analysis of the duration and magnitude of the resulting current reductions. Nanopore in ultrathin SiN membranes, as well as 2D membranes, improve the signal- to-noise ratio for molecular detection and analysis because the resistance to the ionic flow through a nanopore increases linearly with the nanopore thickness, so both the magnitudes of the ionic current and the blocked current with a translocating molecule increase with decreasing nanopore height. Specifically, we seek to make solid-state ionic-current based nanopore sequencing possible by combining several important components: we propose to demonstrate a two-layer on- chip solid-state SiN-2D-pore system that limits the range of DNA motion through two parallel proximal pores that are electrically independently addressable. We create devices containing a second layer with one silicon nitride (SiN) pore, parallel to a primary layer containing the atomically-thin 2D pore that confine ssDNA within a device to a restricted geometry, yet allow the free motion of salt ions to maintain a high signal-to-noise ratio. We propose a specific two-layer concept, where the two layers are in close proximity, with two independent electrical connections, and corresponding chip device architecture to achieve this goal. In this method, there is a central, highly sensitive 2D pore which we refer to as the main sensing/sequencing 2D nanopore. A secondary layer has a second pore sharing the same electrode pair as the sensing pore, but also having its own independent electrode pair to be probed separately. Although we have two pores, they can operate as a continuous system due to their proximity. We outline the 3D finite element analysis modeling and practical implementation (two versions) of these concepts with Si-based technology, including advantages and challenges involved for DNA (and biomolecule) sequencing (analysis) in solution. Our approach eliminates the need for any enzymes and enables DNA and biomolecules to be guided through robust and long-lasting nanopores, facilitated by the custom- designed chip combining the best of what the SiN and 2D pores can currently offer. Illustration 1: Proposed two-layer device concept for this NIH R21 proposal, relying on minimization of DNA entropic motion using two proximal, parallel SiN-2D po...