Nanofabricated Devices for Picoliter-Scale Analyses of Embryonic Gene Regulation

许多重要的生物分子分析研究是不可行的，因为目前的技术太不敏感，规模太大，无法用极小的样本量进行实验。这项提议的总体目标是探索微米和纳米级技术的潜力，以克服这些限制。这项工作的两个基本原则是：分子检测能力可以在微/纳米级设备中得到极大增强。小体积，从微升到皮升或更少，可以更合适地在小型设备中处理。这些努力的最终目标是实现集成仪器，允许从单个细胞中原位提取和分析目标分子成分。在这些最初的努力中，近期的目标是使用为这项研究专门设计的原型微型设备来研究参与海胆胚胎发育过程的一类特定分子。转录因子(TF)蛋白通过与特定DNA序列的高亲和力结合，调节胚胎时间和空间受限区域的差异基因表达。在现有技术下，功能性转铁蛋白/DNA结合研究只能使用数十万个胚胎来实现。为了研究来自胚胎内小区域或单个细胞的TF/DNA结合，提供以下功能的微设备将在本研究计划中依次构建、评估和优化：细胞、小组织片段和溶液的操纵和运输。裂解细胞/细胞器以打开其内容物以供进一步分析。试剂混合，将合成的荧光寡核苷酸和未标记的非特异DNA与细胞提取物结合。皮升溶液的电导率测量/光学吸收光谱。荧光分子的电泳法分离检测。在当代纳米结构研究中广泛使用的许多材料系统被选择来优化电子或光子性能，但这些系统不一定与生物系统理想地匹配，甚至可能是兼容的。这一初始计划的一个重要推动力是开发材料/工艺组合，特别是高度各向异性的干法蚀刻工艺，这种工艺允许在保持生物兼容性的同时进行高分辨率微细加工。器件将使用高分辨率光学和电子束光刻、干法和湿法蚀刻工艺以及热沉积和溅射沉积的薄膜材料来构建。在它们开发的每一步，所创建的仪器将被应用于前述的TF/DNA结合分析。这种在纳米制造和生物方面进行的迭代和互动研究，将推动生物科学新类别的高分辨率仪器的实际实现。预计这项工作将在生物现象科学和生物仪器技术方面产生新的纳米级研究成果。

Many important analytical studies of biomolecules are not feasible because current technology is both too insensitive and at a scale too gross for experimentation with extremely small sample volumes. The overall objective of this proposal is to explore the potential of micro- and nanometer scale technology to overcome these limitations. Two underlying principles motivate this work: molecular detection capability can be greatly enhanced in micro/nanometer scale devices. small volumes, from micro- to pico-liters or less, can be more appropriately handled in small devices. The ultimate objective in these endeavors is to realize integrated instruments that permit in situ extraction and analysis of targeted molecular components from an individual cell. In these initial efforts, the immediate goal is to study a specific class of molecules involved in the process of sea urchin embryogenesis using prototypical microdevices specifically designed for this research. Transcription factor (TF) proteins regulate differential gene expression in temporally and spatially restricted regions of the embryo by binding with high affinity to specific DNA sequences. With existing technology, functional TF/DNA binding studies can only be achieved using hundreds of thousands of embryos. To study TF/DNA binding from small regions within an embryo, or from individual cells, microdevices that provide the following functions will be sequentially constructed, evaluated, and optimized in this research pro~ram: Manipulation and transport of cells, small tissue fragments, and solutions. Lysis of cells/organelles to open their contents for further analysis. Reagent mixing, to combine synthetic, fluorescent oligonucleotides and unlabeled, non-specific DNA with cell extracts. Electrical conductivity measurements/optical absorption spectroscopy upon picoliter solutions. Electrophoretic separation and detection of fluorescent molecules. Many materials systems widely employed in contemporary nanostructure research are chosen to optimize electronic or photonic properties, but these are not necessarily ideally suited, nor perhaps even compatible, with biological systems. An important thrust in this initial program is the development of materials/processing combinations, especially highly-anisotropic dry etching procedures, which permit high resolution microfabrication while retaining biological compatibility. Devices will be constructed using both high resolution optical and electron beam lithography, dry and wet etching processes, and both thermallyand sputter- deposited thin film materials. At each step of their development, the instruments created will be applied to the aforementioned TF/DNA binding assays. This iterative and interactive research, carried out on both nanofabrication and biological fronts, will drive the practical attainment of new classes of high resolution instruments for biological science. It is anticipated that this work will spawn new research efforts at the nanometer scale in both the science of biological phenomena and the technology of bioinstrumentation.