Microfluidic nanoarrays for high-throughput analysis of biological nanostructures

项目总结/摘要 小的膜结合纳米结构在生物学中是普遍存在的。占据亚细胞大小的区域， 生物纳米结构包括细胞器、分泌囊泡、细胞外囊泡，例如外来体， 微泡、凋亡小体和由革兰氏阴性细菌产生的外膜囊泡（OMV）。 此外，亚微米结构如突触体，其是神经元的分离的突触前末梢， 可以从均质化的组织中获得。所有这些结构，即使与单一来源隔离，也可以 在其化学、物理和生理特性方面显示出极大的异质性。生物 长期以来，通过对单个细胞进行测量来分析和解释异质性。持续增 单细胞分析的广泛领域已经使用传统的分析技术，包括分离， 电化学和质谱来揭示单细胞或亚细胞结构的特性， 从传统的批量集合测定中隐藏出来。批量集成分析对异步事件也是盲的 通过单细胞、颗粒或分子研究揭示的。然而，对单细胞、颗粒、 或者分子本质上是低通量的，除非采用某种多路复用策略。成像是一 常见的多路复用方法，然而，它也可以是相对低的吞吐量，除非采取步骤来打包 尽可能多的单个物体。因此，需要新的战略，使高- 对单个生物纳米结构的通量测量，以揭示化学和 生理特性在这里，我们提出了一种高通量微流体纳米阵列方法， 对数百到数万个单个生物纳米结构的单个实体测量 同步我们的平台依赖于分子纳米点的高密度图案化， 专门捕获感兴趣的单个对象。然后将纳米点捕获阵列集成到多通道 微流控装置和单个脂质体、OMV或突触体被纳米点捕获。我们 微流体设计允许在将不同试剂或试剂梯度递送到不同的细胞中的空间选择性 阵列的区域。这种方法适用于几乎任何膜结合的纳米级生物 纳米结构。为了证明该平台的多功能性，它将用于多种不同的检测， 脂质体、OMV和突触体。由于这些测定是在大群个体上进行的， 结构，它们可以照亮化学和生理特性的隐藏分布和异质性， 包括OMV表面上的毒素含量或毒素含量与OMV大小之间的相关性。突触体内 我们将研究突触体内钙动力学，神经递质摄取和释放， 和通过内吞/胞吐的膜循环。

Project Summary / Abstract Small membrane-bound nanostructures are ubiquitous in biology. Occupying the subcellular size regime, biological nanostructures include organelles, secretory vesicles, extracellular vesicles, such as exosomes, microvesicles, apoptotic bodies, and outer membrane vesicles (OMV) produced by Gram-negative bacteria. Additionally, sub-micron structures such as synaptosomes, which are isolated presynaptic terminals of neurons, can be derived from homogenized tissues. All of these structures, even when isolated from a single source, can display an extreme amount of heterogeneity in their chemical, physical, and physiological properties. Biological heterogeneity has long been analyzed and accounted for by making measurements on single cells. Indeed, the broad field of single cell analysis has used traditional analytical techniques, including separations, electrochemistry, and mass spectrometry to reveal properties of single cells or subcellular structures that are hidden from traditional bulk ensemble assays. Bulk ensemble assays are also blind to the asynchronous events that are revealed by single cell, particle, or molecule studies. However, measurements on single cells, particle, or molecules are intrinsically low-throughput unless some sort of multiplexing strategy is employed. Imaging is a common multiplexing approach, however it too can be relatively low-throughput unless steps are taken to pack as many single objects as possible in a field of view. Therefore new strategies are required to make high- throughput measurements on single biological nanostructures to reveal heterogeneities in chemical and physiological properties. Here we propose a high-throughput microfluidic nanoarray approach that facilitates single entity measurements on hundreds to tens of thousands of individual biological nanostructures simultaneously. Our platform relies on ultrahigh density patterning of nanodots of molecules that are used to specifically capture single objects of interest. The nanodot capture arrays are then integrated into multichannel microfluidic devices, and individual liposomes, OMVs, or synaptosomes are captured by the nanodots. Our microfluidic designs allow spatial selectivity in delivery of different reagents or gradients of reagents to different zones of the arrays. This approach is applicable to virtually any membrane-bound nanoscale biological nanostructure. To demonstrate the versatility of this platform, it will be used for a number of different assays on liposomes, OMVs, and synaptosomes. Since these assays are conducted on large groups of individual structures, they can illuminate hidden distributions and heterogeneity of chemical and physiological properties, including toxin content on OMV surfaces or correlation between toxin content and OMV size. In synaptosomes we will examine the heterogeneities in intrasynaptosomal Ca2+ dynamics, neurotransmitter uptake and release, and membrane cycling by endocytosis/exocytosis.