Microsystem-based Formation of Recombinant Protein Fibers

基于微系统的重组蛋白纤维的形成

• 批准号: 7707004
• 负责人: Xiaoyi Wu
• 金额: $ 22.35万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7707004
• 关键词: Affect; Aluminum; Amaze; Amino Acid Sequence; Benign; Characteristics; Controlled Study; Development; Drug Delivery Systems; Duct (organ) structure; Elastin; Engineering; Environmental Risk Factor; Fiber; Goals; Ionic Strengths; Ions; Lead; Length; Liquid substance; Lysine; Mechanics; Methodology; Microfluidic Microchips; Microfluidics; Microscope; Modeling; Molecular; Monitor; Optics; Peptide Sequence Determination; Performance; Physiological; Process; Property; Protein Biosynthesis; Protein Engineering; Proteins; Pump; Recombinant Proteins; Recombinants; Role; Scientist; Side; Silk; Site; Sodium Chloride; Solutions; Solvents; Spiders; Structure; System; Techniques; Technology; Temperature; Tissue Engineering; Water; Width; aqueous; base; cold temperature; crosslink; microsystems; mimicry; new technology; polypeptide; pressure; public health relevance; simulation; tool

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.

描述（由申请人提供）：拟议项目的目标是使用集成微流体装置研究蛋白质纤维的形成，并开发用于生成用于生物医学应用的高性能蛋白质纤维的“绿色”工艺。尽管蛋白质合成和制造技术取得了重大进步，但蜘蛛仍然是在低压、环境温度和以水为溶剂的情况下生产异常坚固的丝纤维的最佳工程师。据推测，蜘蛛可以精细地控制丝蛋白的类型、蛋白质溶液的生理条件以及丝线的机械变形，以纺出强度极高的丝纤维。在这里，我们假设微系统技术的使用为研究丝纤维形成机制以及随后开发生产高性能蛋白质纤维的“绿色”工艺提供了一个重要的范例。具体来说，由丝和弹性蛋白衍生的多肽序列组成的重组丝弹性蛋白样蛋白（SELP）将用作该项目的模型材料。具体目标 1. 确定影响纤维形成和纤维二级结构的蛋白质溶液特性。将制造一个由基于 PDMS 的微通道和铝制微加热器组成的集成微流体系统，用于局部温度控制，用于研究 SELP 纤维的形成。 SELP水溶液将通过主通道被泵入微流系统，而盐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。具体目标 2. 研究决定 SELP 纤维形成、二级结构和机械性能的纺丝管道的流动特性。 SELP 螺纹的机械变形将通过缩小通道宽度和改变流速来控制。由此产生的 SELP 纤维的机械性能和二级结构将被表征。在这项工作中，将利用计算流体动力学 (CFD) 模拟和本构分析模型来增强我们对剪切/拉伸流对纤维形成、结构和性能影响的理解。具体目标 3. 探索蛋白质一级结构对调节 SELP 纤维的形成、结构和特性的影响。将合成由不同长度的丝/弹性蛋白样块和交联位点组成的SELP，并检查它们形成纤维的能力。将研究掺入赖氨酸残基、丝状块的尺寸以及丝与弹性蛋白样块的比率对SELP纤维的形成、结构和性能的影响。 公共健康相关性：基于微系统的重组蛋白纤维的形成蜘蛛在环境温度、低压和水作为溶剂的情况下使用“绿色”工艺生产出异常坚固的丝纤维，这让科学家们感到惊讶。我们建议使用集成微流体装置作为研究丝纤维形成机制和开发“绿色”纤维制造技术的重要工具。值得注意的是，蜘蛛非凡的丝纺过程的揭秘和“绿色”制造技术的发展可能会导致高性能蛋白质纤维的工程应用于许多生物医学应用。