CAREER: Genetic Diagnostics at Surfaces - Designing a Biological Polyelectrolyte System

本研究是对生物聚电解质刷及其与溶液中聚电解质相互作用的基础性研究。此外，这项研究的目的是在生物技术和聚合物科学领域之间建立一座重要的桥梁，聚合物科学领域正在为生物系统提供越来越重要的见解。过去的一项巨大研究工作是研究合成聚合物刷子，这种刷子由一端拴在表面上的聚合物阵列组成。当这些聚合物带电(聚电解质刷子)时，许多基本问题仍然没有答案：理论预测了一个复杂的状态图，但相对较少的实验存在。这里提出的研究通过关注最重要的带电生物聚合物DNA的刷子，促进了对聚电解质刷子的基本理解。将对与游离DNA溶液接触的DNA刷子进行实验研究，重点是测量游离DNA渗透到刷子及其与拴系DNA互补区域的附着(“杂交”)的平衡和动力学方面。理论将同时发展，以接近全面的，基于分子的理解所涉及的机制。如果成功，这项工作将：(I)促进对聚电解质刷子的基本了解，(Ii)揭示为合成聚电解质刷子开发的理论框架可以在多大程度上应用于这一生物聚合物系统，(Iii)建立杂交过程的平衡和动力学如何反映刷子的内部组织，以及(Iv)导致对现代医学和生物技术(如DNA微阵列)功能所涉及的机制的系统理解。主要的实验工具将是共聚焦荧光显微镜，调制椭圆偏振法、X射线光电子能谱和X射线反射率将作为关键的二次表征工具。%DNA刷子是极其重要的生物技术的核心：利用DNA芯片和微阵列处理用于疾病研究和医疗诊断的遗传信息。由于这些方法的工作原理是表面拴系核酸和游离核酸之间的杂交，计划中的研究提供了必要的基本指导，以减少目前在使用和优化它们时的经验主义。该研究计划还与多项教育活动紧密结合，包括：(1)通过为从高中到研究生阶段的学生提供研究机会来刺激智力增长；(2)通过开发一系列高中科学课程的实验单元来与更广泛的教育界建立联系，这些实验单元使用通用的“超市”材料演示科学和工程原理；以及(3)为核心本科生实验室开发新的教学结构，将教会学生通过研究型的、目标驱动的项目进行学习。成功实现拟议计划的研究和教育目标得到了战略伙伴关系的支持，其中包括乔治·华盛顿高中，曼哈顿学区的一所城市学校，以及工业和机构研究合作。

The proposed research is a fundamental investigation of biological polyelectrolyte brushes and their interaction with polyelectrolytes in solution. In addition, the research aims to build an important bridge between biotechnology and the field of polymer science, which is increasingly contributing vital insights to biological systems. A huge past research effort has investigated synthetic polymer brushes, consisting of arrays of polymers tethered by one end to a surface. When these polymers are charged (polyelectrolyte brushes) many basic questions remain unanswered: theory has predicted a complex diagram of states but relatively little experiment exists. The research proposed here advances fundamental understanding of polyelectrolyte brushes by focusing on brushes of the single most important charged biological polymer, DNA. Experimental investigations of DNA brushes placed in contact with free DNA solutions will be carried out, with emphasis on measuring equilibrium and kinetic aspects of the penetration of the free DNA into the brush and its attachment ("hybridization") to complementary regions of the tethered DNA. Theory will be simultaneously developed to approach a comprehensive, molecular-based understanding of the mechanisms involved. If successful, this work will: (i) advance basic understanding of polyelectrolyte brushes, (ii) reveal to what extent theoretical frameworks developed for synthetic polyelectrolyte brushes may be carried over to this biopolymer system, (iii) establish how equilibrium and kinetics of the hybridization process reflect internal organization of the brush, and (iv) result in a systematic understanding of the mechanisms involved in the functioning of modern medical and biological technologies such as DNA microarrays. The primary experimental tool will be confocal fluorescence microscopy, with modulated ellipsometry, X-ray photoelectron spectroscopy, and X-ray reflectivity serving as key secondary characterization tools.%%%DNA brushes are at the heart of a biotechnology of enormous importance: the processing of genetic information for disease studies and medical diagnosis by use of DNA chips and microarrays. Since these methods work on the principle of hybridization between surface-tethered and free nucleic acids, the planned research provides the fundamental guidance needed to reduce the current empiricism in their use and optimization. The research program also closely integrates with multiple educational initiatives, including: (1) stimulating intellectual growth by providing research opportunities for students from high school through graduate levels, (2) forging links with the broader educational community through development of a series of experimental units for high school science courses that demonstrate scientific and engineering principles using common "supermarket" materials, and (3) developing new pedagogical structure for the core undergraduate laboratory that will teach students to learn through research-type, objective-driven projects. Successful accomplishment of the research and educational goals of the proposed plan is supported through strategic partnerships, which include George Washington High School, an urban school in the Manhattan School District, and industrial and institutional research collaborations.