Microsystem-based Formation of Recombinant Protein Fibers

描述(由申请人提供)：该项目的目标是利用集成的微流控装置研究蛋白质纤维的形成，并开发用于生物医学应用的产生高性能蛋白质纤维的“绿色”工艺。尽管在蛋白质合成和制造技术方面取得了重大进步，但蜘蛛仍然是最好的工程师，可以在低压、常温和以水为溶剂的条件下生产出非常坚固的丝纤维。据推测，蜘蛛精细地控制着丝蛋白的类型、蛋白质溶液的生理条件以及丝线的机械变形，以纺制出具有非凡强度的丝纤维。在这里，我们假设，微系统技术的使用为研究蚕丝纤维形成的机制提供了一个重要的范例，并随后开发了生产高性能蛋白质纤维的“绿色”工艺。具体地说，由从蚕丝和弹性蛋白中提取的多肽序列组成的重组真丝-类弹性蛋白(SELP)将被用作拟议项目的模型材料。具体目的1.确定影响纤维形成和纤维二级结构的蛋白质溶液特性。为了研究SELP纤维的形成，将制作一个集成的微流控系统，该系统由基于PDMS的微通道和铝微加热器组成，用于局部温度控制。SELP水溶液将通过主通道被泵入微流控系统，而盐溶液和酸性或碱性溶液将通过侧通道被驱动，以受控的方式调节SELP溶液的离子强度和pH值。PH、离子和温度对纤维形成和纤维二级结构的影响将使用光学显微镜进行监测，并使用各种光谱方法进行表征。具体目的2.研究纺丝管道的流动特性，它决定了SELP的纤维形成、二次结构和机械性能。SELP螺纹的机械变形将通过缩小通道宽度和改变流量来控制。将对所得到的SELP纤维的机械性能和二级结构进行表征。在这项工作中，将利用计算流体力学(CFD)模拟和本构分析模型来加强我们对剪切/拉伸流动对纤维形成、结构和性能的影响的理解。具体目的3.探讨蛋白质一级结构在调节SELP纤维的形成、结构和性质中的作用。将合成由不同长度的丝状/弹性蛋白样块和交联点组成的SELP，并检测它们形成纤维的能力。研究了加入赖氨酸残基、丝状纤维的大小以及丝状纤维与弹性蛋白纤维的比例对SELP纤维的形成、结构和性能的影响。 与公共健康相关：基于微系统的重组蛋白纤维的形成蜘蛛让科学家们感到惊讶，它在常温、低压和以水为溶剂的情况下使用“绿色”工艺生产出异常坚固的丝绸纤维。我们建议使用集成微流控器件作为研究真丝纤维形成机理和开发“绿色”纤维制造技术的重要工具。值得注意的是，蜘蛛非凡的蚕丝纺丝工艺的揭开神秘面纱，以及“绿色”制造技术的发展，可能会导致将高性能蛋白质纤维工程应用于许多生物医学应用。

DESCRIPTION (provided by applicant): The goal of the proposed project is to study protein fiber formation using an integrated microfluidic device, and to develop "green" processes for generating performance protein fibers for biomedical applications. Despite significant advances in protein synthesis and fabrication technology, spiders are still the best engineers producing extraordinarily strong silk fibers at low pressures, ambient temperatures, and with water as solvent. It has been postulated that spiders finely control the types of silk proteins, the physiological conditions of protein solutions, as well as the mechanical deformation of the silk thread in order to spin silk fibers of exceptional strength. Here, we hypothesize that the use of microsystem technology provides an important paradigm for studying the mechanisms involved in silk fiber formation and, subsequently, developing "green" processes for producing performance protein fibers. Specifically, recombinant silk-elastin-like proteins (SELPs) comprised of polypeptide sequences derived from silk and elastin will be used as a model material for the proposed project. Specific Aim 1. Determine the protein solution characteristics that influence the fiber formation and the fiber secondary structures. An integrated microfluidic system consisting of PDMS-based microchannels and an aluminum micro heater, for local temperature control, will be fabricated for studying SELP fiber formation. SELP aqueous solution will be pumped into the microfluidic system through the main channel, while salt and acidic or basic solutions will be driven through side channels to adjust the ionic strength and pH value of the SELP solution in a controlled manner. The effects of pH, ions and temperature on the fiber formation and the fiber secondary structures will be monitored using an optical microscope and characterized using a variety of spectroscopic methodologies. Specific Aim 2. Investigate the flow properties of the spinning duct that determine the fiber formation, secondary structures, and mechanical properties of SELPs. Mechanical deformation of the SELP thread will be controlled by narrowing the channel width and varying the flow rates. The resulting mechanical properties and secondary structures of SELP fibers will be characterized. In this effort, computational fluid dynamics (CFD) simulations and a constitutive analytical model will be utilized to enhance our understanding of the effects of shear/elongational flows on fiber formation, structure, and properties. Specific Aim 3. Explore the effects of the protein primary structures in modulating the formation, structure, and properties of SELP fibers. SELPs composed of varying lengths of silk-/elastin-like blocks and crosslinking sites will be synthesized, and their capability to form fibers will be examined. The influence of incorporating lysine residues, the size of silk-like blocks, as well as the ratio of silk- to elastin- like blocks on the formation, structure, and properties of SELP fibers will be investigated. PUBLIC HEALTH RELEVANCE: Microsystem-Based Formation of Recombinant Protein Fibers Spiders have amazed scientists by producing extraordinarily strong silk fibers using "green" processes at ambient temperatures, low pressures and with water as solvent. We propose to use an integrated microfluidic device as an important tool for studying the formation mechanisms of silk fibers and for developing "green" fiber fabrication techniques. Significantly, the demystification of spiders' remarkable silk spinning process and the development of "green" fabrication techniques may lead to the engineering of performance protein fibers for many biomedical applications.