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: 10452542
• 负责人: Adam Roger Hall
• 金额: $ 30.59万
• 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/10452542
• 关键词: Address; Amplifiers; Biological; Biological Models; Biological Process; Biology; Buffers; Caliber; Cell Line; Characteristics; Charge; Chondroitin Sulfates; Communities; Data; Degenerative polyarthritis; Detection; Development; Devices; Diagnostic; Disease; Disease Progression; Enzyme-Linked Immunosorbent Assay; Event; Exhibits; Foundations; Future; Glycobiology; Glycosaminoglycans; Goals; Heparitin Sulfate; Human; Hyaluronan; Hyaluronic Acid; Hydration status; Immunity; Individual; Inflammation; Joints; Label; 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; Research; Research Personnel; Rheumatoid Arthritis; Rheumatology; Role; Sampling; Severities; Specimen; Staging; Statistical Methods; Structure; Structure-Activity Relationship; Synovial Fluid; System; Techniques; Technology; Testing; Time; Tissues; Urine; Urologic Cancer; Urologic Oncology; Yang; 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）是体内普遍存在的生物分子，其作用从 调节关键免疫调节功能作为滑液的主要润滑成分 （SF）在关节中。因此，该分子的准确和全面表征对 提高我们对广泛的生物过程和疾病状态的理解，并可能有 转化诊断中的潜在下游应用。但是，当前用于评估HA的技术 有重大的局限性。例如，诸如酶联免疫吸附测定法（ELISA）之类的技术是 擅长量化HA，但忽略了使HA分子量的临界结构功能关系 （MW）其角色的定义特征。能够解决HA MW的方法有挑战 包括有限的动态范围（质谱）和较大的样品质量要求（凝胶电泳）， 通常缺乏确定集中度的能力，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-）纳米孔进行稳健的分子 分析。在SS-Nananopore测量中，带电的生物分子通过A运输 合成，纳米尺度孔径。每个人都会产生当前的签名或“事件” 可以测量和解释以表示有关螺纹分子的特征的易位， 包括MW。另外，这些事件的总速率以分子浓度尺度缩放，提供了 通过在解决方案中量化分析物的方法。结果，该平台是唯一的探测HA的位置。在 目的1的目标1，我们将首先通过研究来优化SS-Nananopore设备性能以进行HA分析 关键的实验参数系统地扩展我们的隔离方案，以靶向炎症 - 特别是HA。然后，我们将利用系统的高灵敏度来分析HA 通常在探测中具有挑战性的生物流体。这将通过执行测量来完成 在两个疾病状态下，HA被认为具有特殊相关性：肾脏中的HA 类风湿关节炎中的癌症（AIM 2），血浆和尿HA（AIM 3）。我们假设增加 我们的SS-Nanopore方法提供的敏感性和定量将使HA之间的相关性 丰度/尺寸分布和疾病进展将被识别，并用于微小或无创的 诊断。该项目将由一个研究人员团队进行，该团队将其定位为成功， 具有SS纳米孔分析，分子诊断，糖生物学，统计方法，泌尿外科方面的专业知识 肿瘤学和风湿病学。最终的技术将解决当前分析的挑战 方法，扩大了对HA的考虑及其在基本生物学和疾病中的不同功能。