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

拟议研究的总体目标是使用一种酶？末端脱氧核苷酸转移酶(TDT？)？从含有多种非天然、化学或光学功能核苷酸的表面生长出长的单链(Ss)DNA链？在不断增长的DNA链中，我们将这种技术称为表面引发的DNA酶聚合(SIEP)。这项研究的结果是，它将提供一种新的、通用的芯片上信号检测和放大方案，该方案广泛适用于芯片上传感器、异质免疫分析、蛋白质和DNA微阵列以及microRNA的检测。为了实现所提出的研究目标，将对线形和分支DNA结构的SIEP进行系统的、机理的研究和优化，将一组天然和非天然的核苷酸嵌入到聚合的DNA链中嵌入化学活性核苷酸或光学或电化学反应检测部分。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，这些结构可以用于多功能和多路复用的分子检测方案。这里开发的用于信号放大的技术简单和实验方便的方法将：(1)允许分析稀薄或弱结合的样品，从而提高阵列灵敏度；(2)允许在不牺牲信号强度的情况下减小特征尺寸，这将使更多的信息能够被编码在单个衬底上；以及(3)为护理点诊断的信号检测提供稳健的策略。这项拟议的研究具有变革性，因为它具有广泛的实用价值，因为它将导致一种新型和多功能的检测和放大平台技术的发展，该技术广泛适用于广泛的芯片上传感器、异类免疫分析、蛋白质和DNA微阵列以及新兴的microRNA检测领域。这项研究在教育和外展方面的更广泛影响是：(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)和一名听障学生(通过现有的REU计划)参与团队科学；(2)它将通过杜克大学的女性计划，面向达勒姆公立学校的4-6年级女学生，提供实践实验室经验，培养她们对科学和工程的兴奋和理解；(3)它将通过位于达勒姆的北卡罗来纳科学与数学学院(NCSSM)的导师计划，直接吸引高度积极、精选的高中生参与该项目。

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.