An innovative label-free dual-nanopore TOF sensor for detection and identification of single molecules

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TITLE: Biotechnology Resource Center of BioModular Multi-scale Systems (CBM2) for Precision Medicine TR&D 2: An innovative label-free dual-nanopore TOF sensor for detection and identification of single molecules Abstract Nanofluidic devices offer promising and highly innovative approaches for analyzing single molecules and obtaining biophysical information that cannot be realized using microfluidics due to scaling issues. The ability to provide reliable, rapid, quantitative, and low-cost identification of single molecules will offer exciting new opportunities for a broad range of biomedical applications. The goal of TR&D 2 of the Biotechnology Resource Center of BioModular Multi-scale Systems (CBM2) for Precision Medicine is to produce an innovative label-free nanofluidic sensor for not only detecting single molecules, but identifying them as well. The hypothesis behind our nanosensor is, “individual molecules moving electrokinetically through a 2D nanotube will experience time- of-flight (TOF) that are dependent upon their molecular identity.” We have demonstrated this concept in our active P41 with baseline separations of fluorescently labeled deoxynucleotide, ribonucleotide monophosphates and oligonucleotides via their time-of-flight (TOF) through a polymer-based nano-column. TR&D 2 aims to extend the TOF differentiation of single molecules to a label-free approach. Label-free readout of the molecular- dependent TOF is achieved using dual-nanopore TOF sensors, where two or more in-plane nanopores are placed at either end of a nano-column, which is used for nanoscale electrophoresis. The molecular TOF measured by the time delay between two consecutive current transient signals provides a signature to allow for identification of single molecules. In the active P41 Center, preliminary data have demonstrated this capability. This TR&D will develop the hardware and software required for high throughput label-free TOF sensing. High rate manufacturing of the nanosensor with sub-5 nm in-plane nanopores will be achieved via NIL using the manufacturing protocols that we developed in our active P41. This will be combined with the development of data processing electronics and single-molecule identification algorithms based on machine learning to increase identification accuracies. Technologies required to build multiple nanosensors on a single chip (>100 sensors per chip) will be developed, which include large area molding tools, replication processes, reliable electrical/fluidic connections, and electronics/software. This will allow for high throughput processing of single molecules. As a demonstration of the technology developed in this TR&D, we will use the dual-nanopore TOF sensor to assess epigenetic modifications of DNA. By integrating solid-state nano-reactors (TR&D 1) with optimized dimensions of the in-plane nanopores and nano-columns, this sensor can be configured to provide molecular information from unamplified targets (DNA, RNA, and proteins) with unprecedented identification accuracies (>95%). This will transform single-molecule processing to allow servicing a broad biomedical community for a wide range of applications.