Technologies for analysis of biological molecules and particles are crucial for advancing research in biology and health care. There is a significant gap in technology for rapid, label-free characterization of unknown particles and molecules in the size range of 10-500 nm. While larger particles and cells are easily characterized by a variety of techniques including Coulter counters and cytometers, inadequate signal-to-noise ratio is a key limitation of detecting smaller particles using Coulter counters. Advances in nanotechnology have resulted in nanopore sensors based on the Coulter counter principle, which are being developed to a large extent for DNA sequencing. Their sensitivity arises due to their size being comparable to the size of the analyte. However, these sensors are typically not suitable for analysis of larger particles or molecules with varying unknown size, and their fabrication is also difficult. A common feature of nanopores and Coulter counters is that the analyte particle or molecule escapes into the solution after measurement, which limits the time during which the particle is analyzed and results in poor signal-to-noise ratio. However, if multiple measurements were possible on the same particle, the signal-to-noise ratio may be expected to be dramatically enhanced due to statistical averaging over the measurements. The goal of this project is to develop a nanofluidic Coulter counter system with feedback control that will enable multiple measurements on the same particle, thereby greatly improving the signal to noise ratio for sizing nanoscale analytes (Aim 1). This system will be evaluated for its ability to size fragments of genomic DNA (Aim 2) and to distinguish between nanospheres with slight differences in size as models for biological particles (Aim 3). This goal will be achieved using a system consisting of a nanochannel (50-500 nm) flanked by two nanofluidic reservoirs that serve to trap analytes and prevent their escape during measurement. The devices will be fabricated using soft lithography techniques that are amenable to integration into fluidic systems. The proposed device is the first step towards integrated systems capable of manipulation, processing, and analysis of single particles and molecules at the nanoscale. If successful, this project will lay the foundations for integrated fluidic systems for analysis of single particles, PCR-free assays, and may eventually enable simple biochemical analyses such as DNA and protein digestion and fingerprinting at the single molecule level. PUBLIC HEALTH RELEVANCE: Narrative In this project we will develop a nanofluidic system for sizing of large DNA fragments with a nanochannel sensor. This work may result in significant improvement in signal-to-noise ratios for label-free analysis of single biological particles and molecules in the nanoscale size range.