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学，它可以在典型的患者/提供者交互的15分钟时间范围内，在护理点准确和定量地测量多个蛋白质生物标记物。具体地说，我们建议在我们的E-AB传感平台中进行重大技术进步，以实现这一目标。E-AB传感器是基于电极结合的DNA适配子的结合诱导折叠。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(几秒到几分钟)，并具有足够的选择性，可以直接用于尿液、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、低容量(亚微升)和完全电子化(电化学)的，因此很可能适用于护理点应用。支持这一建议的关键假设是，随着灵敏度和检测置信度的充分提高，我们可以调整E-AB传感器平台，从临床样本中提供可用于治疗的诊断信息。虽然我们的长期目标是制造适用于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、具有代表性的应用上，这一应用很容易与标准的临床方法进行比较：测定尿液和血清趋化因子，以诊断急性肾移植排斥反应。为此，我们建议改进E-AB传感器的灵敏度和样品间的稳定性，使其达到常规临床使用所需的检测极限和重复性。我们将通过1)选择更高亲和力的适配子(通过一种新颖、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)开发能够实现精确背景减除和校正的控制传感元件来实现这些目标。我们组建了一支创新和协作的团队，在高通量适配子选择、电化学检测和临床实践方面拥有丰富的专业知识。我们相信，该项目的成功将产生重大影响--它将代表着朝着简单、无试剂、低成本的诊断平台迈出了一大步，该平台广泛适用于护理点的分子诊断。 公共卫生相关性：目前检测诊断蛋白的方法需要繁琐、资源密集的实验室程序，通常需要几小时或几天才能为临床医生提供治疗上可操作的诊断信息。在这里，我们建议开发一种新型的电子(电化学)生物传感器，旨在快速(&lt；15分钟)、直接在未经处理的临床样本(即尿液和指枪血液样本)中定量多个蛋白质生物标志物。这项技术的成功实施将大大缩短检查和治疗之间的时间，这反过来将提高医疗效率并改变医疗成本结构。虽然我们的长期目标是开发适用于各种诊断蛋白中任何一种的检测设备，但我们最初的努力将集中在一个具有代表性的、特别紧迫的应用上，该应用很容易实现与标准临床方法的比较：尿液和血液中趋化因子的测定，以诊断肾移植排斥反应。

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.