Nanoscale Optofluidic Pathogen Detection

描述(由申请人提供)：需要快速诊断新出现的病毒威胁，以及将单个或多个点多态与疾病状态和药物反应联系起来的可能性，这导致了最近对开发新的高通量核酸生物传感器的兴趣。在这一应用中，R21的支持被要求用于“纳米级光流体传感器阵列”的开发，该阵列代表了高保真、高通量和无标记生物传感的新范式。这项技术依赖于将流体系统缩小到与光的波长相同的尺度，并利用独特的硅纳米光子结构的性质来访问逝去的场并提供反应地点的空间局部化。正如这项技术所证明的那样：(1)具有全谱水平的检测灵敏度而不需要靶标记，(2)能够以亚微米的间距独立地实现单个纳米传感位点的功能化(3)确保每个溶液相核酸靶有多个机会与其表面固定的补体杂交(4)允许固有的二维多路复用和(5)通过独特的电动严格技术加强反应的特异性。虽然广泛适用于广泛的核酸应用，但在这里，我们建议通过对一系列独立样本的同时多路复用的病毒病原体(重点是被疾病控制和预防中心确认为本世纪新兴疾病之一的四种登革病毒血清型)的特定检测来演示该平台。这项探索性的工作建立在PI和Co-PI在集成微流控器件、纳米级光流控集成、高通量核酸筛选和基于RNA的病毒病原体生物传感器方面的背景下。在这项工作中，我们将专注于传感器平台的实验开发，为R01应用做准备，该设备将应用于临床诊断，并将包括传感器分析之前的样品准备步骤，如免疫磁分离、RNA提取和预浓缩。登革热病毒是热带和亚热带地区的主要公共卫生问题。据估计，每年有5000万人死于登革出血热，其中包括25万-50万例登革出血热，死亡率为5%-10%。纳米级光流体传感器阵列的开发可以提供一种技术，通过该技术，可以以高度平行的形式以非常低的数量检测像登革热这样的病毒病原体，但不限于登革热。近期的主要应用将是作为早期检测平台。

DESCRIPTION (provided by applicant): The need to rapidly diagnose emerging viral threats along with the potential for associating individual or multiple point polymorphisms with disease states and pharmacological responses has lead to a recent interest in the development of new high-throughput nucleic acid biosensors. In this application, R21 support is requested for the development of "Nanoscale Optofluidic Sensor Arrays" which represent a new paradigm in high fidelity, high throughput, and unlabeled biosensing. The technique relies on shrinking the fluidic system down to the same scale as the wavelength of light and using the properties of a unique silicon nanophotonic structure to gain access to the evanescent field and to provide spatial localization of the reaction site. As is demonstrated this technique: (1) has attogram level detection sensitivity without the need for target labeling, (2) enables independent functionalization of individual nano-sensing sites with sub-micron spacing (3) ensures each solution phase nucleic acid target has multiple opportunities to hybridize with its surface immobilized complement (4) allows for inherent two dimensional multiplexing and (5) enforces reaction specificity through a unique electrokinetic stringency technique. Though broadly applicable to a wide range of nucleic acid applications, here we propose to demonstrate the platform through the specific detection of viral pathogens (focusing on the four serotypes of Dengue virus which has been identified by the Center of Disease Control and Prevention as one of the emerging diseases of our century) multiplexed simultaneously against a series of independent samples. This exploratory work builds on the PI's and Co-PI's background in integrated microfluidic devices, nanoscale optofluidic integration, high-throughput nucleic acid screening and RNA based viral pathogen biosensors. In this work we will focus on the experimental development of the sensor platform in preparation for an R01 application in which the device will be applied to clinical diagnostics and will incorporate sample preparation steps such as immunomagnetic separation, RNA extraction and pre- concentration prior to sensor analysis. Dengue virus is a major public health concern in tropical and subtropical areas. It causes an estimated 50 million illnesses annually, including 250,000-500,000 cases of Dengue Hemorrhagic Fever with 5-10% of mortality. Development of the Nanoscale Optofluidic Sensor Array could provide a technique by which viral pathogens like, but not limited to Dengue, can be detected at very low quantities in a highly parallel format. The near term major application will be as an early stage detection platform.