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

项目摘要 我们正在开发微型和纳米流体设备来探测病毒衣壳、细菌和细胞外小泡 在单粒子水平上，提高了空间和时间分辨率。单粒子(或数字) 测量不仅提高了敏感度，还提供了对种群异质性的洞察。信息 通过以高通量、多路传输的格式执行这些分析来进一步增强内容，其中 跟踪个别事件，但也获得总体统计数据。我们的目标是罕见或罕见的 事件，这些事件可以显著影响系统的整体功能或命运，因为这些事件通常 在批量样品上进行测量时被遮挡。在第一个项目中，我们正在研究病毒衣壳 装配和拆卸。为了监测衣壳的反应，我们正在设计面内纳米流体装置 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、无损的施胶 技术。阻性脉冲传感可实时检测事件，并具有足够的灵敏度来监控 在生物相关浓度和一系列反应条件下组装。有了这些 纳米流体设备，我们正在评估组装效应器和杂波对组装过程的影响 并产生各种粒子形态，包括动力学捕获的中间体和反常粒子 结构。在第二个项目中，我们用微流控装置跟踪细菌的发育和老化。 它们集成了纳米通道阵列来物理捕获细菌。纳米通道限制了细胞的生长 细菌在一个维度上，当与荧光显微镜结合时，图像分析从 一个从三维到一维的问题，大大简化了。生长和分裂速率，亚细胞 功能、表观遗传效应和抗生素反应很容易被跟踪更长的时间和 跨越多代人。在第三个项目中，我们正在分析来自血清、尿液、 和腹水，以及它们的细胞外小泡。为了对这些多糖进行彻底的表征，我们 结合化学标记策略中和和区分唾液酸键异构体 微流控毛细管电泳质谱联用。多聚糖的差异 它们的大小、岩藻糖化程度和唾液酸化程度以及唾液酰键异构体的比例都是定量的。 样本。我们正在使用单粒子技术来表征分子的物理和化学性质 胞外小泡将这些特性与其糖链图谱相关联。

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.