Rapid detection of diagnostic chemokines

描述（由申请人提供）：在这里，我们建议开发分子诊断，可以在典型的患者/提供者交互的15分钟时间框架内，在护理点准确定量地测量多种蛋白质生物标志物。具体来说，我们提出了E-AB传感平台的主要技术进步来实现这一目标。E-AB传感器是基于结合诱导折叠的电极结合DNA适体。重要的初步数据表明，E-AB平台是敏感的（纳摩尔到几十皮摩尔），快速（秒到分钟），和选择性足够直接用于尿液，血清和其他复杂的，未经处理的临床材料。E-AB传感器同样是无试剂，可重复使用，小体积（亚微升）和全电子（电化学），因此可能适用于护理点应用。该提议的关键假设是，通过充分提高灵敏度和检测置信度，我们可以调整E-AB传感器平台，从临床样本中提供治疗上可操作的诊断信息。虽然我们的长期目标是制造适合检测多种诊断蛋白的E-AB传感器，但我们最初的努力将集中在一个特定的、有代表性的应用上，与标准的临床方法进行比较是很容易实现的：尿和血清趋化因子的测量，诊断急性肾移植排斥反应的发作。为此，我们建议在此改进E-AB传感器的灵敏度和样品对样品的稳定性，使其达到常规临床使用所需的检测限和再现性。我们将通过以下方式实现这些目标：1)选择更高亲和力的适配体（通过一种新颖的、超高效的微流体- SELEX技术），2)显著提高E-AB增益（通过优化探针密度和几何形状、氧化还原和附着化学以及其他重要的制造变量），以及3)开发控制传感元件，实现精确的背景减除和校正。我们已经组建了一支创新和协作的团队，在高通量适配体选择，电化学检测和临床实践方面具有重要的专业知识。我们相信，该项目的成功将产生重大影响——它将代表着向简单、无试剂、低成本的诊断平台迈出一大步，该平台广泛适用于护理点的分子诊断。

DESCRIPTION (provided by applicant): Here we propose the development of molecular diagnostics that can accurately and quantitatively measure multiple protein biomarkers at the point-of-care, within the 15-minute timeframe of a typical patient/provider interaction. Specifically, we propose major technological advancements in our E-AB sensing platform to achieve this goal. The E-AB sensor is based on the binding-induced folding of electrode- bound DNA aptamers. Significant preliminary data indicates that the E-AB platform is sensitive (nanomolar to tens of picomolar), rapid (seconds to minutes), and selective enough to employ directly in urine, serum and other complex, unprocessed clinical materials. E-AB sensors are likewise reagentless, reusable, low volume (sub-microliter) and fully electronic (electrochemical), and are thus likely adaptable to point-of-care applications. The key hypothesis underlying this proposal is that, with sufficiently improved sensitivity and detection confidence, we can adapt the E-AB sensor platform to provide therapeutically actionable diagnostic information from clinical samples. And while our long-term objective is the fabrication of E-AB sensors suitable for the detection of a wide range of diagnostic proteins, our initial efforts will focus on a specific, representative application for which comparison with standard clinical approaches is readily achieved: the measurement of urinary and serum chemokines diagnostic of episodes of acute renal allograft rejection. Toward this end, we propose here improvements in the sensitivity and sample-to-sample stability of the E-AB sensors such that they reach the detection limits and reproducibility required for routine clinical use. We will achieve these goals by 1) selection of higher affinity aptamers (via a novel, ultra-efficient microfluidic- SELEX technology), 2) significant improvements in E-AB gain (via optimization of probe density and geometry, redox and attachment chemistry, and other important fabrication variables), and 3) development of control sensing elements that will enable precise background subtraction and correction. We have assembled an innovative and collaborative team with significant expertise in high-throughput aptamer selection, electrochemical detection and clinical practice. We believe that the success of this project will have significant ramifications -it will represent a large step toward a simple, reagentless, low cost diagnostic platform that is broadly applicable to molecular diagnostics at the point of care.