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

用于透明质酸丰度和尺寸分布平移分析的固态纳米孔

• 批准号: 10693188
• 负责人: Adam Roger Hall
• 金额: $ 30.58万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-05 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10693188
• 关键词: Address; Amplifiers; Assessment tool; Biological; Biological Models; Biological Process; Biology; Buffers; Cell Line; Characteristics; Charge; Chondroitin Sulfates; Communication; Communities; Data; Degenerative polyarthritis; Detection; Development; Devices; Diagnostic; Diameter; Disease; Disease Progression; Enzyme-Linked Immunosorbent Assay; Event; Exhibits; Future; Glycobiology; Glycosaminoglycans; Goals; Heparitin Sulfate; Human; Hyaluronan; Hyaluronic Acid; Hydration status; Immunity; Individual; Inflammation; Joints; Label; Letters; Liquid substance; Lubrication; Mass Spectrum Analysis; Measurement; Measures; Methods; Molecular; Molecular Analysis; Molecular Weight; Nature; Patients; Performance; Physiological; Plasma; Polymers; Polysaccharides; Positioning Attribute; Protocols documentation; Renal carcinoma; Reporting; Research; Research Personnel; Rheumatoid Arthritis; Rheumatology; Role; Sampling; Severities; Specimen; Staging; Statistical Methods; Structure; Structure-Activity Relationship; Synovial Fluid; System; Techniques; Technology; Technology Assessment; Testing; Time; Tissues; Urine; Urologic Cancer; Urologic Oncology; analytical method; angiogenesis; biological systems; cell motility; cohort; cost; frontier; gel electrophoresis; human subject; immunoregulation; improved; in vivo; insight; kidney cell; molecular diagnostics; molecular marker; nano; nanopore; nanoscale; noninvasive diagnosis; prognostic; prognostic value; single molecule; solid state; sugar; tool; translational diagnostics; urinary

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.

项目摘要 透明质酸(或透明质酸，HA)是体内普遍存在的一种生物分子，其作用范围从 调节关键免疫调节功能成为滑液的主要润滑成分 (Sf)在关节中。因此，对分子的准确和全面的表征对于 提高我们对广泛的生物过程和疾病状态的理解，并可能 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，像酶联免疫吸附试验(ELISA)这样的技术是 擅长对HA进行量化，但忽略了使HA相对分子质量增加的关键结构-功能关系 (Mw)其作用的决定性特征。能够解决HA MW的方法面临以下挑战 包括有限动态范围(质谱仪)和大样本质量要求(凝胶电泳)， 通常缺乏确定浓度的能力，需要多种技术才能完成 评估。为了解决这一差距，我们建议使用固态(SS-)纳米孔来构建强大的分子 分析。在SS纳米孔测量中，带电的生物分子通过一个 人工合成的纳米级光圈。每个人都会产生一个当前的签名或‘事件’ 可被测量和解释以表示有关穿线分子的特征的易位， 包括MW。此外，这些事件的总发生率随分子浓度而变化，从而提供了 对溶液中的分析物进行定量的方法。因此，该平台在探测HA方面具有独特的定位。在……里面 本项目的目标1，我们将首先通过研究来优化用于HA分析的SS-Nanore装置的性能 关键实验参数系统化，并扩展我们的分离方案，以也针对炎症- 特别标明HA。然后，我们将利用我们的系统的高灵敏度来分析HA 传统上很难探测的生物流体。这将通过执行测量来实现 在两种疾病状态下，HA被认为具有特殊的相关性：肾脏中的尿液HA 癌症(目标2)和类风湿关节炎的血浆和尿液透明质酸(目标3)。我们假设增加的 我们的SS-纳米孔方法提供的灵敏度和定量将使HA之间的相关性 确定丰度/大小分布和疾病进展，并用于微创或非侵袭 诊断。这个项目将由一组研究人员进行，他们的定位独特，能够成功， 在SS纳米孔分析、分子诊断学、糖生物学、统计学方法、泌尿学方面拥有专业知识 肿瘤学和风湿学。由此产生的技术将解决当前分析技术的挑战 方法：拓宽对HA及其在基础生物学和疾病中的多种功能的认识。