Along with nucleic acids and proteins, glycosaminoglycans (GAGs) are considered a foundational building block of biology. However, unlike their more widely-studied counterparts, the tools for analyzing GAGs are relatively lacking. In our prior R01, we developed solid-state (SS-) nanopore technology for GAG analysis, focusing on the key molecule hyaluronan (or hyaluronic acid, HA) and establishing a method for determining molecular weight (MW) distribution from a small sample amenable to physiological levels. This has allowed new investigations into the size-function relationship of HA. The focus of this project will be to increase the value of the SS-nanopore platform both in terms of HA measurement and as a general GAG analytical tool. In the first Aim, we will first implement a strategy for HA quantification that can be coupled with our existing MW assessment to render a more versatile technology. HA abundance is an independent identified biomarker in several disease states, making it valuable in combination with size distribution. Then, we will establish a microfluidic infrastructure to enable HA extraction from biological matrices and subsequent SS-nanopore analysis to be performed in a self-contained format, improving the accessibility of the approach to other laboratories. In the second Aim, we will first perform pilot measurements toward an improved reagent for extraction of HA coupled to protein markers of inflammation (heavy chains), with the goal of targeting a single protein isoform and establishing feasibility for selection between the multiple known isoforms that are thought to have independent functions. Finally, we will extend the SS-nanopore analytical approach to other GAGs, focusing on the important molecules heparan sulfate and chondroitin sulfate and integrating methods for assessing sulfation levels. Overall, this project will result in a more powerful technology that will further the field of glycobiology. 2.