Nanoscale Optofluidic Pathogen Detection

描述（由申请人提供）：快速诊断新兴病毒威胁的需要沿着将个体或多点多态性与疾病状态和药理学反应相关联的潜力，导致最近对开发新的高通量核酸生物传感器的兴趣。在这项申请中，R21支持要求开发“纳米级光流体传感器阵列”，这代表了高保真度，高通量和无标记生物传感的新范例。该技术依赖于将流体系统缩小到与光的波长相同的尺度，并使用独特的硅纳米光子结构的特性来获得对倏逝场的访问并提供反应位点的空间定位。正如所证明的，这种技术：（1）具有attogram级别的检测灵敏度，而不需要目标标记，（2）能够以亚微米间距独立功能化各个纳米传感位点（3）确保每个溶液相核酸靶具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性技术加强反应特异性。虽然广泛适用于广泛的核酸应用，在这里，我们提出通过特异性检测病毒病原体（重点是登革热病毒的四种血清型，其已被疾病控制和预防中心确定为我们世纪的新兴疾病之一），同时对一系列独立样品进行多重检测来证明该平台。这项探索性工作建立在PI和Co-PI在集成微流体设备，纳米级光流体集成，高通量核酸筛选和基于RNA的病毒病原体生物传感器的背景之上。在这项工作中，我们将专注于传感器平台的实验开发，为R 01应用做准备，其中该设备将应用于临床诊断，并将包括样品制备步骤，如免疫磁性分离，RNA提取和传感器分析前的预浓缩。登革热病毒是热带和亚热带地区的一个主要公共卫生问题。据估计，它每年造成5000万人患病，其中包括25万至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量检测病毒病原体，如但不限于登革热。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.