Electrochemical arrays for the detection of small molecule drugs

概述：用于检测DNA和RNA的微阵列已经建立，蛋白质微阵列正在迅速发展。相比之下，用于小分子定量检测的阵列的发展明显滞后。在这里，我们提出了一种无试剂的电化学方法，用于平行实时监测血液和其他复杂介质中的一组小分子，这一进步将被证明在从诊断到药物发现到基础生物科学（例如代谢组学）的应用中具有重要的实用性。我们的方法是基于电化学的、基于适配体（E-AB）的平台，该平台敏感、快速、无试剂、选择性强，足以直接用于血清和其他大部分未经处理的生物材料。虽然我们的长期目标是制造用于有效实时监测任何小分子的E-AB设备，但拟议的研究侧重于滥用药物和氨基糖苷类抗生素的特定高价值示例。无试剂E-AB平台利用电化学来监测电极结合适体的靶诱导折叠。迄今为止，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，每种传感器都是敏感的（微摩尔到皮摩尔的检测限），并且有足够的选择性，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，实时检测分析物流中的小分子，如流动的血清。为了支持这种新兴的方法，拟议的研究计划侧重于自上而下的开发E-AB阵列，用于同时实时定量小分子药物。实现这一目标将需要开发改进方案，以选择针对小分子的高亲和力，高特异性DNA适体，选择新的适体及其在高增益，高灵敏度E-AB传感器中的适应性，进一步改进E-AB平台本身，最后，制造和测试微米级E-AB阵列，这些进步将牢固地建立这种潜在的有前途的通用传感技术的实用性。用于同时检测多种rna的微阵列已经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-蛋白质相互作用的理解。一种定量的、可并行的监测小分子的方法可能会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证明同样具有变革性。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色谱或竞争分析，这些技术不适合快速、平行的分析物检测。在这里，我们提出了一种基于适体体的电化学（E-AB）技术的发展，用于血液和其他复杂介质中多种小分子分析物的并行、实时定量。虽然我们的长期目标是开发适用于任何小分子连续、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上，作为高价值的、临床相关的靶标，以开发这种有潜力的通用传感技术。

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.