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.
Designing a portable, rapid, inexpensive, sensitive and selective field-usable RNA sensing platform would have a significant impact in clinical diagnostics, recreation water monitoring, global health care, anti-terrorism/ biowarfare and food safety.
