Project Summary Hyaluronan (or hyaluronic acid, HA) is a ubiquitous biomolecule in vivo, with diverse roles ranging from regulating key immunomodulatory functions to serving as the primary lubricating component of synovial fluid (SF) in joints. Consequently, the accurate and comprehensive characterization of the molecule is critical to improving our understanding of a broad range of biological processes and disease states, and may have potential downstream applications in translational diagnostics. However, current technologies for assessing HA have significant limitations. For example, techniques like the enzyme-linked immunosorbent assay (ELISA) are adept at quantifying HA but ignore the critical structure-function relationship that makes HA molecular weight (MW) a defining characteristic of its role. Approaches that are able to resolve HA MW have challenges that include limited dynamic range (mass spectrometry) and large sample mass requirement (gel electrophoresis), and generally lack the ability to determine concentration, necessitating multiple techniques for complete assessment. To address this gap, we propose to employ solid-state (SS-) nanopores for robust molecular analysis. In a SS-nanopore measurement, charged biomolecules are transported electrically through a synthetic, nanometer-scale aperture. A current signature, or `event', is produced with each individual translocation that can be measured and interpreted to denote characteristics about the threading molecule, including MW. In addition, the overall rate of these events scales with molecular concentration, providing a means by which to quantify analytes in solution. As a result, the platform is uniquely positioned to probe HA. In Aim 1 of this project, we will first optimize SS-nanopore device performance for HA analysis by investigating key experimental parameters systematically and expand our isolation protocols to also target inflammation- marked HA specifically. Then, we will take advantage of the high sensitivity of our system to analyze HA in biofluids that are conventionally challenging to probe. This will be accomplished by performing measurements in the context of two disease states where HA is thought to have particular relevance: urinary HA in kidney cancer (Aim 2) and plasma and urinary HA in rheumatoid arthritis (Aim 3). We hypothesize that the increased sensitivity and quantitation offered by our SS-nanopore approach will enable correlations between HA abundance/size distribution and disease progression to be identified and used for minimally- or non-invasive diagnostics. This project will be conducted by a team of researchers that is positioned uniquely to succeed, with expertise in SS-nanopore analysis, molecular diagnostics, glycobiology, statistical methods, urological oncology, and rheumatology. The resulting technology will address the challenges of current analytical methods, widening consideration of HA and its varied functions in basic biology and disease.

R01GM134226

2020-09-05

2024-06-30