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

项目摘要 我们正在开发微和纳米流体设备，以探测病毒衣壳，细菌和细胞外囊泡 在单粒子水平上具有改进的空间和时间分辨率。单粒子（或数字） 测量不仅提供了改进的灵敏度，而且还提供了对群体异质性的洞察。信息 通过以高通量、多路复用的形式进行这些测定， 跟踪单个事件，但也获得总体统计数据。我们的目标是罕见的 事件，这可能会对系统的整体功能或命运产生重大影响，因为这些事件通常 当对批量样品进行测量时会被掩盖。在第一个项目中，我们正在研究病毒衣壳 组装和拆卸。为了监测与衣壳的反应，我们正在设计面内纳米流体装置， 具有串联的多个纳米孔，用于连续脉冲传感，这是一种无标记的非破坏性尺寸测量， 法电阻脉冲感应可真实的检测事件，并具有足够的灵敏度进行监控 在生物学相关浓度和反应条件范围内组装。与这些 纳米流体设备，我们正在评估如何组装效应器和离液剂影响组装过程 并产生各种颗粒形态，包括动力学捕获的中间体和异常的 结构.在第二个项目中，我们正在使用微流体设备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 细菌在一个维度上，当与荧光显微镜相结合时，图像分析从 一个三维到一维的问题，并大大简化。生长和分裂率，亚细胞 功能、表观遗传效应和抗生素反应很容易长时间跟踪， 跨越几代人。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囊泡。为了彻底表征这些聚糖，我们 将化学标记策略与分析相结合以中和和区分唾液酸连接异构体， 微流控毛细管电泳和毛细管电泳-质谱。聚糖差异 在这些实验中定量了岩藻糖基化和唾液酸化的大小、程度以及唾液酸连接异构体的比率。 样品我们正在使用单粒子技术来表征的物理和化学性质， 细胞外囊泡，以将这些性质与其聚糖谱相关联。

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.