Microfluidic nanoarrays for high-throughput analysis of biological nanostructures

项目概要/摘要 小型膜结合纳米结构在生物学中无处不在。占据亚细胞大小范围， 生物纳米结构包括细胞器、分泌囊泡、细胞外囊泡，例如外泌体， 革兰氏阴性菌产生的微泡、凋亡小体和外膜泡 (OMV)。 此外，亚微米结构，例如突触体，是神经元的孤立突触前末端， 可以源自匀浆组织。所有这些结构，即使与单一来源隔离，也可以 它们的化学、物理和生理特性表现出极大的异质性。生物的 长期以来，人们一直通过对单细胞进行测量来分析和解释异质性。确实， 单细胞分析的广泛领域使用了传统的分析技术，包括分离、 电化学和质谱法揭示单细胞或亚细胞结构的特性 隐藏在传统的批量集成分析中。批量集成分析也对异步事件视而不见 通过单细胞、颗粒或分子研究揭示的。然而，对单细胞、粒子、 或分子本质上是低通量的，除非采用某种多重策略。成像是一个 常见的多路复用方法，但是它的吞吐量也可能相对较低，除非采取步骤进行打包 视野中尽可能多的单个物体。因此需要新的策略来实现高 对单个生物纳米结构进行通量测量，以揭示化学和纳米结构的异质性 生理特性。在这里，我们提出了一种高通量微流控纳米阵列方法，该方法有助于 对数百到数万个单独的生物纳米结构进行单一实体测量 同时。我们的平台依赖于分子纳米点的超高密度图案，这些纳米点用于 专门捕捉单个感兴趣的对象。然后将纳米点捕获阵列集成到多通道中 微流体装置和单个脂质体、OMV 或突触体被纳米点捕获。我们的 微流体设计允许在将不同试剂或试剂梯度输送到不同的区域时具有空间选择性。 阵列的区域。该方法几乎适用于任何膜结合纳米级生物 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体的个体上进行的 结构，它们可以阐明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。在突触体中 我们将检查突触体内 Ca2 动力学、神经递质摄取和释放的异质性， 和通过胞吞作用/胞吐作用进行膜循环。

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.