Microfluidic nanoarrays for high-throughput analysis of biological nanostructures

项目摘要/摘要 微小的膜结合纳米结构在生物学中普遍存在。占据着亚细胞大小的区域， 生物纳米结构包括细胞器、分泌小泡、细胞外小泡，如外切体， 革兰氏阴性杆菌产生的微泡、凋亡体和外膜小泡(OMV)。 此外，亚微米结构，如突触体，是孤立的神经元突触前终末， 可以从均质的组织中衍生出来。所有这些结构，即使与单一来源隔离，也可以 在化学、物理和生理特性上表现出极大的异质性。生物学 长期以来，通过对单个细胞进行测量来分析和解释异质性。事实上， 单细胞分析的广泛领域使用了传统的分析技术，包括分离， 电化学和质谱学，以揭示单细胞或亚细胞结构的性质 隐藏在传统的整体分析中。批量集成分析也对异步事件视而不见 通过单细胞、粒子或分子研究揭示的。然而，对单个细胞、粒子、 或者分子本质上是低吞吐量的，除非采用某种多路复用策略。成像是一种 常见的多路传输方法，但是，除非采取打包步骤，否则它也可能是相对较低的吞吐量 视场中尽可能多的单个对象。因此，需要新的战略来使高效率。 对单个生物纳米结构的吞吐量测量以揭示化学和化学的异质性 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 对数百到数万个单个生物纳米结构的单一实体测量 同时。我们的平台依赖于分子纳米点的超高密度图案，用于 专门捕捉感兴趣的单个对象。然后将纳米点捕获阵列集成到多通道中 微流控装置和单个脂质体、OMV或突触体被纳米点捕获。我们的 微流控设计允许空间选择性地将不同试剂或试剂梯度输送到不同的 阵列的区域。这种方法几乎适用于任何膜结合的纳米生物 纳米结构。为了展示该平台的多功能性，它将用于多个不同的分析 脂质体、OMV和突触体。因为这些化验是在一大群个体身上进行的 结构，它们可以阐明化学和生理性质的隐藏分布和异质性， 包括OMV表面的毒素含量或毒素含量与OMV大小的关系。在突触体内 我们将研究突触体内钙动力学、神经递质摄取和释放、 以及内吞/胞吐作用的膜循环。

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.