Rapid detection of diagnostic chemokines

描述（由申请人提供）：在这里，我们提出了分子诊断的发展，可以准确和定量测量多种蛋白质生物标志物在床旁，在15分钟的时间范围内的一个典型的患者/提供者的互动。具体来说，我们建议在我们的E-AB传感平台上实现重大技术进步，以实现这一目标。E-AB传感器基于电极结合的DNA适体的结合诱导的折叠。重要的初步数据表明，E-AB平台灵敏（纳摩尔至数十皮摩尔）、快速（数秒至数分钟），并且具有足够的选择性，可直接用于尿液、血清和其他复杂的未加工临床材料。E-AB传感器同样是无试剂、可重复使用、低体积（亚微升）和全电子（电化学）的，因此可能适用于护理点应用。该提议的关键假设是，在充分提高灵敏度和检测置信度的情况下，我们可以调整E-AB传感器平台，以提供来自临床样本的治疗上可行的诊断信息。虽然我们的长期目标是制造适用于检测各种诊断蛋白质的E-AB传感器，但我们最初的努力将集中在一个特定的、具有代表性的应用上，与标准临床方法进行比较很容易实现：测量诊断急性肾移植排斥反应的尿液和血清趋化因子。为此，我们在此提出改进E-AB传感器的灵敏度和样品间稳定性，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体（通过新型的超高效微流体- SELEX技术），2）显著改善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）开发能够实现精确背景扣除和校正的控制传感元件。我们已经组建了一支创新和协作的团队，在高通量适体选择，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-它将代表向简单，无试剂，低成本诊断平台迈出的一大步，该平台广泛适用于护理点的分子诊断。 公共卫生相关性：目前用于检测诊断蛋白的方法需要繁琐的、资源密集型的实验室程序，其通常需要数小时或数天来为临床医生提供治疗上可行的诊断信息。在这里，我们提出开发一类新的电子（电化学）生物传感器，其旨在直接在未处理的临床样品中快速（<15分钟）、即时定量多种蛋白质生物标志物（即，尿液和手指采血管样本）。这种技术的成功实施将大大缩短检查和治疗之间的时间，从而提高医疗保健效率并改变医疗保健成本结构。虽然我们的长期目标是开发适用于检测各种诊断蛋白的设备，但我们的初步努力将集中在一个具有代表性且特别紧迫的应用上，与标准临床方法进行比较很容易实现：测量诊断肾移植排斥的尿和血液中的趋化因子。

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. PUBLIC HEALTH RELEVANCE: Current methods for the detection of diagnostic proteins require cumbersome, resource-intensive laboratory procedures that typically require hours or days to provide clinicians with therapeutically actionable diagnostic information. Here we propose the development of a new class of electronic (electrochemical) biosensors aimed at the rapid (<15 minutes), point-of-care quantification of multiple protein biomarkers directly in unprocessed clinical samples (i.e., urine and finger-lance samples of blood). The successful implementation of such a technology would significantly shorten the time between examination and treatment, which, in turn, will improve healthcare efficacy and transform the healthcare cost structure. While our long-term objective is the development of devices suitable for the detection of any of a wide range of diagnostic proteins, our initial efforts will focus on a representative and particularly pressing application for which comparison with standard clinical approaches is readily achieved: the measurement of urinary and blood-borne chemokines diagnostic of kidney transplant rejection.