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: 10255500
• 负责人: Adam Roger Hall
• 金额: $ 30.62万
• 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/10255500
• 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.

项目概要 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： 调节关键的免疫调节功能，作为滑液的主要润滑成分 （SF）在关节中。因此，准确而全面的分子表征对于 提高我们对广泛的生物过程和疾病状态的理解，并且可能 转化诊断中的潜在下游应用。然而，目前评估 HA 的技术 有很大的局限性。例如，酶联免疫吸附测定 (ELISA) 等技术 擅长定量 HA，但忽略了影响 HA 分子量的关键结构-功能关系 (MW) 其作用的一个决定性特征。能够解决 HA MW 问题的方法面临以下挑战： 包括有限的动态范围（质谱）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 评估。为了解决这一差距，我们建议采用固态（SS-）纳米孔来实现稳健的分子 分析。在 SS 纳米孔测量中，带电生物分子通过 合成的纳米级孔径。每个人都会产生当前的签名或“事件” 可以测量和解释易位以表示有关线程分子的特征， 包括兆瓦。此外，这些事件的总体发生率与分子浓度成正比，从而提供了 定量溶液中分析物的方法。因此，该平台具有独特的定位来探测 HA。在 该项目的目标 1，我们将首先通过调查优化 SS-纳米孔装置的 HA 分析性能 系统地研究关键实验参数，并将我们的隔离方案扩展到也针对炎症- 专门标记了HA。然后，我们将利用我们系统的高灵敏度来分析HA 通常难以探测的生物流体。这将通过执行测量来完成 在两种疾病状态下，HA 被认为具有特别的相关性： 肾脏中的尿 HA 癌症（目标 2）以及类风湿性关节炎中的血浆和尿 HA（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能够实现 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入性治疗 诊断。该项目将由一组研究人员进行，该团队具有独特的成功优势， 拥有 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。由此产生的技术将解决当前分析的挑战 方法，扩大对 HA 及其在基础生物学和疾病中的各种功能的考虑。