Electrochemical arrays for the detection of small molecule drugs

描述(申请人提供)：用于检测小分子药物的电化学阵列摘要：用于检测DNA和RNA的微阵列已经建立得很好，而蛋白质微阵列正在迅速发展。相比之下，用于定量检测小分子的阵列的开发明显滞后。在这里，我们提出了一种无试剂的电化学方法，用于实时并行监测血液和其他复杂介质中的一组小分子，这一进展应被证明在从诊断到药物发现到基础生物科学(如代谢组学)的应用中具有重要的实用价值。我们的方法是基于电化学、适体为基础的(E-AB)平台，该平台具有灵敏、快速、无试剂和足够的选择性，可以直接用于血清和其他主要未经处理的生物材料。虽然我们的长期目标是制造E-AB设备，以有效地实时监测任何小分子，但拟议的研究重点是滥用药物和氨基糖苷类抗生素的具体、高价值的例子。无试剂E-AB平台利用电化学来监测目标诱导的电极结合适体的折叠。到目前为止，我们已经制造了针对蛋白质、小分子和无机离子目标的E-AB传感器，每个目标都是灵敏的(微摩尔对皮摩尔的检测极限)和足够的选择性，可以直接部署在血清、唾液和土壤提取物中。此外，我们的传感器稳定且平衡迅速，允许连续、实时地检测分析物流中的小分子，如流动的血清。为了支持这一新方法，拟议的研究计划将重点放在自上而下开发的E-AB阵列上，旨在同时、实时地定量小分子药物。要实现这一点，需要开发针对小分子的高亲和力、高特异性DNA适配子的改进方案，新适配子的选择及其在高增益、高灵敏度E-AB传感器中的适应性，E-AB平台本身的进一步改进，以及微米级E-AB阵列的制造和测试，这些进展将稳固地确立这一潜在的、有前景的通用传感技术的实用性。同时检测多个RNA的微阵列彻底改变了我们对转录和基因调控的理解，蛋白质阵列正在迅速提高我们对翻译调控和蛋白质相互作用的理解。在包括基础生物科学(如代谢组学)、诊断和药物发现在内的应用中，一种定量、可并行化的小分子监测手段可能会被证明具有类似的变革性。然而，目前检测血液中药物、代谢物和其他小分子的方法通常仅限于层析或竞争分析，这些技术不适合快速、并行的分析物检测。在这里，我们建议开发一种基于电化学适体(E-AB)的技术，用于在血液和其他复杂介质中并行、实时地定量多个小分子分析物。虽然我们的长期目标是开发适合于对任何小分子进行连续、实时监测的E-AB阵列，但这里提出的努力将集中在滥用药物和氨基糖苷类抗生素作为高价值、临床相关的目标，以开发这一潜在的、有前景的通用传感技术。

DESCRIPTION (provided by applicant): Electrochemical arrays for the detection of small molecule drugs Summary: Microarrays for the detection of DNA and RNA are well established, and protein microarrays are advancing rapidly. The development of arrays for the quantitative detection of small molecules, in contrast, has lagged significantly. Here we propose a reagentless, electrochemical method for monitoring a panel of small molecules in parallel, in real time, in blood and other complex media, an advance that should prove of significant utility in applications ranging from diagnostics through drug discovery to the basic biological sciences (e.g., metabolomics). Our approach is based on an electrochemical, aptamer-based (E-AB) platform that is sensitive, rapid, reagentless, and selective enough to employ directly in serum and other largely unprocessed biological materials. And while our long-term objective is the fabrication of E-AB devices for the real-time monitoring of effectively any small molecule, the proposed research focuses on specific, high-value examples of the drugs of abuse and aminoglycosidic antibiotics. The reagentless E-AB platform utilizes electrochemistry to monitor the target-induced folding of an electrode-bound aptamer. To date we have fabricated E-AB sensors against protein, small molecule and inorganic ion targets, each of which is sensitive (micromolar to picomolar detection limits) and selective enough to deploy directly in blood serum, saliva, and soil extracts. Moreover, our sensors are stable and equilibrate rapidly, allowing for the continuous, real-time detection of small molecules in an analyte stream, such as flowing blood serum. In support of this nascent approach, the proposed research program focuses on the top-to-bottom development E-AB arrays directed at the simultaneous, real-time quantification of suites of small-molecule drugs. Achieving this will require the development of improved schemes for the selection of high affinity, high-specificity DNA aptamers against small molecules, the selection of new aptamers and their adaptation in high gain, high- sensitivity E-AB sensors, further improvements in the E-AB platform itself and, finally, the fabrication and testing of micron-scale E-AB arrays, advances that will firmly establish the utility of this potentially promising and general sensing technology.Relevance. Microarrays for the simultaneous detection of multiple RNAs have revolutionized our understanding of transcription and gene regulation, and protein arrays are rapidly improving our understanding of translational regulation and protein-protein interactions. A quantitative, parallelizable means of monitoring small molecules would likely prove similarly transformative in applications including the basic biological sciences (e.g., metabolomics), diagnostics and drug discovery. Current methods for the detection of drugs, metabolites and other small molecules in blood, however, are generally limited to chromatographic or competition assays, techniques that are ill suited for rapid, parallel analyte detection. Here we propose the development of an electrochemical, aptamer-based (E-AB) technology for the quantification of multiple small molecule analytes in parallel, in real time, in blood and other complex media. And while our long-term objective is the development of E-AB arrays suitable for the continuous, real-time monitoring of effectively any small molecule, the efforts proposed here will focus on the drugs of abuse and the aminoglycosidic antibiotics as high-value, clinically relevant targets with which to develop this potentially promising and general sensing technology.