Single-Particle Analysis of Virus Capsids, Bacteria, and Extracellular Vesicles

病毒衣壳、细菌和细胞外囊泡的单粒子分析

• 批准号: 10206640
• 负责人: Stephen C Jacobson
• 金额: $ 62.64万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10206640
• 关键词: 3-Dimensional; Aging; Antibiotics; Ascites; Bacteria; Biological; Biological Assay; Biological Process; Capillary Electrophoresis; Capsid; Chemicals; Coupled; Development; Devices; Dimensions; Epigenetic Process; Event; Fluorescence Microscopy; Generations; Growth; Image Analysis; Individual; Isomerism; Kinetics; Label; Liquid substance; Mass Spectrum Analysis; Measurement; Methods; Microfluidic Analytical Techniques; Microfluidic Microchips; Monitor; Morphology; Physiologic pulse; Polysaccharides; Population; Population Heterogeneity; Population Statistics; Process; Property; Reaction; Sampling; Series; Serum; Structure; System; Techniques; Time; Urine; Virus; chemical property; design; digital; extracellular vesicles; improved; insight; instrumentation; interest; nanochannel; nanofluidic; nanopore; particle; physical property; response; sialylation; temporal measurement

Project Summary We are developing micro- and nanofluidic devices to probe virus capsids, bacteria, and extracellular vesicles at the single-particle level with improved spatial and temporal resolution. Single-particle (or digital) measurements provide not only improved sensitivity but also insight into population heterogeneity. Information content is further enhanced by performing these assays in a high-throughput, multiplexed format, where individual events are tracked, but population statistics are also obtained. We are targeting rare or infrequent events, which can significantly impact the overall function or fate of a system, because these events are often obscured when measurements are made on bulk samples. In the first project, we are studying virus capsid assembly and disassembly. To monitor reactions with capsids, we are designing in-plane nanofluidic devices with multiple nanopores in series for resistive-pulse sensing, which is a label-free, nondestructive sizing technique. Resistive-pulse sensing detects events in real time and has sufficient sensitivity to monitor assembly at biologically relevant concentrations and over a range of reaction conditions. With these nanofluidic devices, we are evaluating how assembly effectors and chaotropes impact the assembly process and produce a variety of particle morphologies, including kinetically trapped intermediates and aberrant structures. In the second project, we are tracking development and aging of bacteria with microfluidic devices that have integrated nanochannel arrays to physically trap bacteria. The nanochannels confine growth of bacteria in one dimension, and when coupled with fluorescence microscopy, image analysis is reduced from a three-dimensional to one-dimensional problem and greatly simplified. Growth and division rates, subcellular functions, epigenetic effects, and antibiotic response are easily tracked for extended periods of time and across multiple generations. In the third project, we are profiling N- and O-glycans derived from serum, urine, and ascites fluid, and their extracellular vesicles. For thorough characterization of these glycans, we are combining chemical labeling strategies to neutralize and differentiate sialyl linkage isomers with analysis by microfluidic capillary electrophoresis and capillary electrophoresis-mass spectrometry. Differences in glycan sizes, degrees of fucosylation and sialylation, and ratios of sialyl linkage isomers are quantified in these samples. We are using single-particle techniques to characterize the physical and chemical properties of extracellular vesicles to correlate these properties with their glycan profiles.

项目摘要 我们正在开发微流体和纳米流体设备，以探测病毒衣壳，细菌和细胞外囊泡 在单粒子水平上，空间和时间分辨率得到改善。单粒子（或数字） 测量不仅可以提高灵敏度，还提供了对种群异质性的洞察力。信息 通过以高通量的多重格式执行这些测定，进一步增强了内容 跟踪个别事件，但还获得了人口统计。我们的目标是稀有或罕见 事件，可能会显着影响系统的整体功能或命运，因为这些事件通常是 在批量样品上进行测量时被遮盖。在第一个项目中，我们正在研究病毒capsid 组装和拆卸。为了监视带状钉的反应，我们正在设计平面纳米流体设备 具有串联的多个纳米孔，用于电阻脉冲传感，这是一种无标签的无损尺寸 技术。电阻脉冲传感实时检测事件，并且具有足够的灵敏度来监测 在生物学相关浓度和一系列反应条件下组装。与这些 纳米流体设备，我们正在评估组装效应子和交际如何影响组装过程 并产生各种粒子形态，包括动力学捕获的中间体和异常 结构。在第二个项目中，我们正在跟踪细菌使用微流体设备的开发和衰老 将纳米渠道阵列整合到物理​​捕获细菌中。纳米通道限制了 细菌在一个维度中，当与荧光显微镜结合时，图像分析从 对一维问题的三维，并大大简化了。增长和分裂率，亚细胞 在长时间的时间内很容易跟踪功能，表观效果和抗生素反应 跨多代。在第三个项目中，我们正在分析源自血清，尿液， 和腹水流体及其细胞外囊泡。为了彻底表征这些聚糖，我们是 结合化学标记策略，以中和和区分siALyl链接异构体和分析 微流体毛细血管电泳和毛细管电泳质量光谱法。聚糖的差异 在这些尺寸，葡萄糖基化和溶解度的程度以及siAllyl链接异构体的比率在其中量化 样品。我们正在使用单粒子技术来表征 细胞外囊泡将这些特性与它们的聚糖谱相关。