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Platforms for the detection of small molecules, proteins and nucleic acids.

机译：用于检测小分子，蛋白质和核酸的平台。

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摘要

The Plaxco laboratory has developed a new class of reagentless, electrochemical sensors that make use of electrode-bound, redox-tagged oligonucleotide probes as their sensing elements. To date, these electrochemical DNA (E-DNA) and electrochemical, aptamer-based (E-AB) sensors can detect specific DNA and RNA sequences, proteins, small molecules and inorganic ions. However, this first generation of devices had low specificity against single nucleotide polymorphisms and the inability to detect targets that do not specifically interact with oligonucleotides. This dissertation describes several improvements to this E-DNA platform via the development of new strategies that address these drawbacks.;We developed and characterized an electrode-bound DNA pseudoknot architecture that enables "signal-on" DNA sensing and increased mismatch detection capabilities. The pseudoknot consists of two stem-loop structures such that, in the absence of target, they hold a redox tag away from the electrode surface, minimizing electron transfer. Upon target binding, the pseudoknot structure is disrupted, liberating a single stranded DNA region, allowing the redox tag to collide with the electrode surface and transfer electrons, thus leading to an increase in signal. This sensor is sensitive (nanomolar detection limits), able to function in complex samples (e.g., blood serum) and, because of the improved thermodynamics of this system, can discriminate single nucleotide polymorphisms.;Many biomolecules can bind to smaller molecules with high affinity. We exploited this observation to develop a new class of sensors. Conjugation of a small molecule recognition element to a DNA scaffold allows biorecognition of a desired target. Upon target binding, the dynamics of the scaffold change, producing a measurable signal change. Detection of several protein targets has been shown with nanomolar detection limits, rapid kinetics and significant selectivity in complex media such as blood serum and environmental samples. Those sensors are also amenable to a competition assay configuration, and thus can be used for the detection of small molecules in solution.
机译：Plaxco实验室开发了新型的无试剂电化学传感器，该传感器利用电极结合的，氧化还原标记的寡核苷酸探针作为其传感元件。迄今为止，这些电化学DNA（E-DNA）和基于适体的电化学（E-AB）传感器可以检测特定的DNA和RNA序列，蛋白质，小分子和无机离子。但是，第一代设备对单核苷酸多态性的特异性低，并且无法检测与寡核苷酸没有特异性相互作用的靶标。本文通过解决这些缺点的新策略，描述了对该E-DNA平台的一些改进。假结由两个茎环结构组成，因此，在没有靶标的情况下，它们使氧化还原标签远离电极表面，从而最大程度地减少了电子转移。靶结合后，假结结构被破坏，释放出单链DNA区域，使氧化还原标签与电极表面碰撞并转移电子，从而导致信号增加。该传感器灵敏（纳摩尔检出限），能够在复杂样品（例如血清）中起作用，并且由于该系统的热力学提高，可以区分单核苷酸多态性。许多生物分子可以高亲和力与较小的分子结合。我们利用这一观察结果开发了新型传感器。小分子识别元件与DNA支架的缀合允许所需靶标的生物识别。在靶标结合时，支架的动力学改变，产生可测量的信号改变。在纳摩尔浓度的检测限，快速动力学和在诸如血清和环境样品等复杂培养基中的显着选择性方面，已显示出对几种蛋白质目标的检测。这些传感器也适用于竞争测定配置，因此可用于检测溶液中的小分子。

著录项

  • 作者

    Cash, Kevin Joseph.;

  • 作者单位

    University of California, Santa Barbara.;

  • 授予单位 University of California, Santa Barbara.;
  • 学科 Chemistry Biochemistry.;Engineering Chemical.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 125 p.
  • 总页数 125
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

  • 入库时间 2022-08-17 11:38:19

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