Solid-state nanopores for translational analysis of hyaluronan abundance and size distribution

项目摘要 透明质酸（或透明质酸，HA）是体内普遍存在的生物分子，具有多种作用， 调节关键免疫调节功能，作为滑液的主要润滑成分 (SF)在关节。因此，分子的准确和全面表征对于 提高了我们对广泛的生物过程和疾病状态的理解， 在翻译诊断中的潜在下游应用。然而，目前用于评估HA的技术 有很大的局限性。例如，如酶联免疫吸附测定（ELISA）的技术被广泛使用。 擅长定量HA，但忽略了使HA分子量 (MW)这是其角色的一个决定性特征。能够解决HA MW的方法具有挑战性， 包括有限动态范围（质谱法）和大样品质量要求（凝胶电泳）， 并且通常缺乏确定浓度的能力，需要多种技术来完成 考核为了解决这一差距，我们提出采用固态（SS-）纳米孔来进行稳健的分子组装。 分析.在SS-纳米孔测量中，带电生物分子通过纳米孔电传输。 合成的纳米级孔径当前签名或“事件”由每个个体产生， 可以被测量和解释为表示关于穿线分子的特征的易位， 包括MW。此外，这些事件的总体发生率与分子浓度成比例，提供了一个 用于定量溶液中分析物的方法。因此，该平台被独特地定位以探测HA。在 本项目的目标1，我们将首先通过调查优化用于HA分析的SS-纳米孔设备性能， 关键的实验参数，并将我们的分离方案扩展到也针对炎症- 特别标记了HA。然后，我们将利用我们系统的高灵敏度来分析HA， 传统上难以探测的生物流体。这将通过执行测量来实现 在HA被认为具有特别相关性的两种疾病状态的背景下：肾脏中的尿HA 癌症（Aim 2）和类风湿性关节炎（Aim 3）中的血浆和尿HA。我们假设， 我们的SS纳米孔方法提供的灵敏度和定量将使HA之间具有相关性 丰度/大小分布和疾病进展，并用于微创或非侵入性 诊断这个项目将由一个研究团队进行，这个团队具有独特的成功优势， 具有SS纳米孔分析，分子诊断，糖生物学，统计方法，泌尿学 肿瘤学和风湿病学。由此产生的技术将解决当前分析的挑战， 方法，拓宽了对HA及其在基础生物学和疾病中的多种功能的考虑。

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.