Develop a Multi-Target Sensor to Detect Dengue Serotypes from Infected Mosquitoes

描述（申请人提供）：登革热是一种由埃及伊蚊传播的全球性流行病。病毒学监测包括监测人和蚊子中的登革热病毒感染，已被用作预测疫情的预警系统。开发一种具有成本效益的即时诊断系统，用于快速，灵敏地检测野外捕获的蚊子中的登革热病毒，这是一项迫切需要，因为它提供了一种可靠的工具来了解自然界中传播的病毒，并有助于设计控制策略。此外，同样重要的是，这样的平台具有识别和区分登革热病毒的四种不同血清型的能力。基于聚合酶链反应（PCR）的检测系统，专门用于实验室条件下。然而，它们的复杂性、使用困难和高成本限制了它们的实用性和在第一反应者和公共卫生官员中的广泛使用。或者，基于登革病毒的直接RNA检测的系统具有鉴定特异性血清型以及比免疫测定快速检测无症状患者中的感染的潜力。如果有足够的灵敏度，这样的系统可以量化病毒载量，这些信息对于做出明智的治疗决定是有用的。在成功完善分子诊断平台的基础上，一个由微流体、登革热病毒培养和RNA分离以及生物传感领域的学术研究人员组成的跨学科团队将合作开发第一个便携式多靶点RNA诊断系统。所提出的系统将不需要PCR扩增或荧光标记，但将允许快速、灵敏（pM浓度）和选择性（单碱基 错配辨别）在45分钟内检测RNA，这在RNA的降解半衰期内。这种努力已经通过微流体在利用离子交换纳米膜中的电动效应用于RNA预浓缩或检测方面的进步而实现。这种方法是创新的，因为我们利用了膜的敏感离子电流响应的变化，表面电荷由于杂交的目标RNA分子。代替电极上的电子转移，传感机制仅依赖于跨纳米膜的离子电流，减轻了由氧化还原和表面剂引起的稳定性问题。此外，我们还建议开发一种芯片上的RNA捕获单元，使用磁珠直接从感染的蚊子中分离RNA。该平台的实现主要有两个目标：（1）利用纳米膜传感器检测登革4种血清型RNA，并优化传感器的灵敏度和选择性;（2）开发检测登革热血清型的多靶点传感器，并整合RNA前体，在一个实施方案中，该方法包括从登革感染的蚊子中分离浓缩膜以及上游RNA捕获单元。该RNA检测平台的成功开发将为其他RNA病毒、DNA病毒（通过简单地取代分子探针）或其他含有RNA的病原体（如细菌甚至寄生虫）的检测带来显著的益处。除了这里介绍的现场检测应用之外，在传染病、病媒和水媒疾病以及食品安全等领域，公共卫生效益相当可观。

DESCRIPTION (provided by applicant): Dengue fever is a great global health epidemic spread by Aedes aegypti mosquitoes. Virologic surveillance, which involves monitoring of dengue virus infection in humans and mosquitoes, has been used as an early warning system to predict an outbreak. Development of a cost-effective point-of-care diagnostic system for rapid, sensitive detection of dengue virus in field-caught mosquitoes is an immediate necessity as it provides a reliable tool to comprehend the virus circulating in nature, and helps in the designing of control strategies. In addition, it is also important that such a platform have the capability to identify and distinguish between the four different serotypes of dengue virus. Polymerase chain reaction (PCR) -based detection systems specifically used under laboratory conditions. However, their complexity, difficulty of use and high cost limit their utility and widespread use among first responders, and public health officials. Alternatively, systems based upon direct RNA detection of dengue virus have the potential to identify the specific serotypes as well as quickly detect infection in asymptomatic patients than immunoassays. Given sufficient sensitivity, such systems can quantify the viral load, information that is useful for making informed therapeutic decisions. Based upon successful efforts at refining a molecular diagnostic platform, an interdisciplinary team of academic researchers in microfluidics, dengue virus culture and RNA isolation, and biosensing will collaborate to develop the first portable and multi-target RNA diagnostic system. The proposed system will not require PCR amplification or fluorescent labeling, yet will allow rapid, sensitive (pM concentration) and selective (single base mismatch discrimination) detection of RNA within 45 minutes which is within the degradation half-life of RNA. This effort has been enabled by microfluidic advances in utilizing the electrokinetic effects in ion- exchange nanomembranes for either RNA pre-concentration or detection. The approach is innovative because we take advantage of the membrane's sensitive ion-current response to the change in surface charge due to the hybridization of target RNA molecules. Instead of electron transfer on electrodes, the sensing mechanism solely relies on ion current across nanomembranes, alleviating the stability issues induced by the redox and surface agents. In addition, we also propose to develop an on-chip RNA capturing unit using magnetic beads to directly isolate RNA from infected mosquitoes. The proposed platform will be realized by pursuing two specific aims: (1) Test serotype-specific oligo probes for detection of al four serotypes of dengue RNA using nanomembrane sensor and optimize the sensor's sensitivity and selectivity performance; (2) Develop a multi- target sensor for detection of all for dengue serotypes and integrate a RNA pre-concentration membrane as well as an upstream RNA capturing unit from dengue infected mosquitoes. The successful development of this RNA detection platform will extend significant benefit to the detection of other RNA viruses, DNA viruses (by simply substituting the molecular probes) or other pathogens containing RNA, such as bacteria or even parasites. Beyond field-detection applications presented here, the public health benefits are considerable in areas such as infectious disease, vector- and water-borne diseases and food safety.