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Kinetics of charge transport through self-assembled DNA monolayers: Influence of DNA defects and applications for biosensing.

机译：电荷通过自组装DNA单层传输的动力学：DNA缺陷的影响和在生物传感中的应用。

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摘要

Charge transport (CT) through DNA is not yet well understood, but is of importance both fundamentally and in applications such as biosensing. The kinetics of charge transport through self-assembled DNA monolayers was examined using the electrochemical techniques of cyclic voltammetry (CV) and square wave voltammetry (SWV) as complimentary methods for verification of extracted kinetic parameters. DNA, synthesized with and without defects to the base pair pi-stack, was attached to gold working electrodes through incorporation of a six-carbon thiolated linker. Electrical properties were monitored by following the redox signal of Nile blue covalently attached to a modified thymine. Controlled variation in temperature revealed from CV and SWV that DNA CT increases dramatically with temperature until the dehybridization of the DNA causes the single strand containing the redox probe to no longer be coupled to the monolayer. 17-mer DNA containing a single CA mismatch was compared directly to fully well-matched DNA of otherwise identical sequence using multiplexed chips. The mismatch defect was found to greatly attenuate CT at low temperatures and gradually approximate a well-matched sequence as temperature increased. In contrast, DNA containing a single missing base (abasic) site showed only marginal CT decrease relative to well-matched DNA at low temperature, but diverged from the well-matched DNA as temperature increased. Experimental data acquired via SWV was fit to theoretical curves using a modified Nelder-Mead simplex algorithm and kinetic parameters were determined. The electron transfer rate constant k0 was compared across different types of DNA monolayers and Arrhenius behavior was observed. CT implications to disruptions in the pi-stack were then utilized for the sensing of enzymes formamidopyrimidine DNA glycosylase (FPG) and uracil-DNA glycosylase (UDG) by incorporation of defects 8-oxoguanine and uracil into DNA monolayers. Sensitive and selective detection of both enzymes was achieved. Additionally, signal loss due to specific enzyme binding activities was monitored in real-time to determine temperature dependent kinetics of enzyme reactions with target DNA substrates. Versatility of the DNA-mediated electrochemical sensing platform was demonstrated through detection of FPG on gold-coated polyacrylonitrile (PAN) nanofibers. These results are important for the improved understanding of DNA CT properties and to the field of biosensing.
机译：通过DNA进行电荷传输（CT）的方法尚不十分清楚，但从根本上讲以及在诸如生物传感等应用中都很重要。通过循环伏安法（CV）和方波伏安法（SWV）的电化学技术，作为验证提取的动力学参数的补充方法，对通过自组装DNA单分子层的电荷传输动力学进行了研究。通过掺入六碳硫醇化连接子将合成的，有缺陷且无缺陷的碱基对pi栈合成的DNA连接到金工作电极上。通过跟踪共价连接到修饰的胸腺嘧啶的尼罗蓝的氧化还原信号来监测电性能。从CV和SWV可以看出，温度的受控变化表明DNA CT随温度急剧增加，直到DNA的去杂化导致包含氧化还原探针的单链不再与单层偶联。使用多重芯片将含有单个CA错配的17-mer DNA直接与其他序列相同的完全匹配的DNA进行比较。发现失配缺陷会在低温下大大衰减CT，并随着温度升高逐渐接近匹配良好的序列。相反，在低温下，相对于匹配良好的DNA，仅包含一个缺失碱基（碱基）位点的DNA仅显示出少量的CT下降，但随着温度升高，与匹配良好的DNA偏离。通过修改的Nelder-Mead单纯形算法，将通过SWV获得的实验数据拟合到理论曲线，并确定动力学参数。比较了不同类型的DNA单层电子传输速率常数k0，观察到了Arrhenius行为。然后，通过将缺陷8-氧代鸟嘌呤和尿嘧啶掺入DNA单层中，利用CT对pi堆栈破坏的影响来检测甲酰胺嘧啶DNA糖基化酶（FPG）和尿嘧啶DNA糖基化酶（UDG）的酶。实现了对两种酶的灵敏和选择性检测。另外，由于特异性酶结合活性而引起的信号损失被实时监测，以确定与靶DNA底物的酶反应的温度依赖性动力学。通过在包金的聚丙烯腈（PAN）纳米纤维上检测FPG，证明了DNA介导的电化学传感平台的多功能性。这些结果对于增进对DNA CT特性的了解以及对生物传感领域而言都是重要的。

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  • 作者

    McWilliams, Marc Allen.;

  • 作者单位

    The University of Texas at Dallas.;

  • 授予单位 The University of Texas at Dallas.;
  • 学科 Biophysics.
  • 学位 Ph.D.
  • 年度 2015
  • 页码 143 p.
  • 总页数 143
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 康复医学;
  • 关键词

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