Surface-Initiated Enzymatic Polymerization of DNA Nanostrutcures for Highly Amplified Sensing

用于高度放大传感的 DNA 纳米结构的表面引发酶聚合

• 批准号: 1033621
• 负责人: Ashutosh Chilkoti
• 金额: $ 36万
• 依托单位: Duke University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1033621&HistoricalAwards=false
• 关键词: Surface; Initiated; Enzymatic; Polymerization; DNA

The overall goal of the proposed research is to use an enzyme ?terminal deoxynucleotidyl transferase (TdT)? to grow long single stranded (ss) DNA chains from a surface that incorporate a diverse range of unnatural ?chemically or optically functional nucleotides? in the growing DNA chain; we call this technology surface initiated enzymatic polymerization (SIEP) of DNA. The outcome of this research is that it will provide a new and versatile, on-chip signal detection and amplification scheme that is broadly applicable to on-chip sensors, heterogeneous immunoassays, protein and DNA microarrays, and for the detection of microRNA. To fulfill the goal of the proposed research, a systematic, mechanistic investigation and optimization of SIEP of linear and branched DNA structures will be carried out with a set of natural and unnatural nucleotides that embed chemically reactive nucleotides, or optically or electrochemically reactive detection moieties into the polymerized DNA chains. The intellectual merit of the research is that it will yield rationally designed, DNA-based nanostructures that can be employed in versatile and multiplexed molecular detection schemes. The technically simple and experimentally convenient approaches for signal amplification developed herein will: (1) allow analysis of dilute or weakly binding samples and thus increase array sensitivity; (2) allow reduction of the feature size without sacrificing signal intensity, which would enable more information to be encoded on a single substrate; and (3) provide robust strategies for signal detection for point-of-care diagnostics. The proposed research is transformative because of its broad utility, as it will lead to the development of a novel and versatile detection and amplification platform technology that is broadly applicable to a broad range of on-chip sensors, heterogeneous immunoassays, protein and DNA microarrays, and for the emerging field of microRNA detection. The broader impact of this research in terms of education and outreach is that: (1) it will engage two PhD students, a Duke undergraduate student (through the Pratt fellows program) and a hearing-impaired student (through an existing REU program) to engage in team science; (2) it will target 4th-6th grade female students from Durham public schools through the FEMMES program at Duke University, by providing hands-on laboratory experiences to foster their excitement and understanding of science and engineering; (3) it will engage highly motivated, selected senior high school students in the project directly through the mentorship program of the North Carolina School of Science and Mathematics (NCSSM) in Durham.

这项研究的总体目标是使用一种酶？末端脱氧核苷酸转移酶（TdT）？从一个表面生长出长的单链DNA链，并结合各种各样的非天然的？化学或光学功能的核苷酸？在不断增长的DNA链中;我们称这种技术为DNA的表面引发酶聚合（SIEP）。这项研究的结果是，它将提供一个新的和多功能的，芯片上的信号检测和放大方案，广泛适用于芯片上的传感器，异质性免疫测定，蛋白质和DNA微阵列，并检测microRNA。为了实现所提出的研究的目标，将用一组天然和非天然核苷酸进行线性和分支DNA结构的SIEP的系统的、机械的研究和优化，所述天然和非天然核苷酸将化学反应性核苷酸或光学或电化学反应性检测部分嵌入聚合的DNA链中。这项研究的智力价值在于，它将产生合理设计的、基于DNA的纳米结构，这些纳米结构可用于多功能和多路复用的分子检测方案。本文开发的用于信号放大的技术上简单且实验上方便的方法将：（1）允许分析稀释或弱结合的样品，从而增加阵列灵敏度;（2）允许减小特征尺寸而不牺牲信号强度，这将使得更多信息能够在单个基底上编码;以及（3）提供用于即时诊断的信号检测的稳健策略。拟议的研究是变革性的，因为它的广泛实用性，因为它将导致一种新的和多功能的检测和扩增平台技术的发展，广泛适用于广泛的芯片上传感器，异质性免疫测定，蛋白质和DNA微阵列，并为microRNA检测的新兴领域。 这项研究在教育和推广方面的广泛影响是：（1）它将吸引两名博士生，一名杜克大学本科生（通过普拉特研究员计划）和一名听力受损的学生（通过现有的REU计划）从事团队科学;（2）它将通过杜克大学的FEMMES项目，针对达勒姆公立学校的4 - 6年级女学生，通过提供动手实验室经验，以促进他们对科学和工程的兴奋和理解;（3）它将通过达勒姆的北卡罗来纳州科学与数学学院（NCSSM）的导师计划，直接吸引积极性高的高中生参与该项目。