PA21259, SBIR, Phase I, Aptamer-upconverting nanocrystal conjugate lateral flow assay for immediate Ebola virus detection

Ebola virus, listed under Category A priority pathogen by NIAID, is one of the most deadly viruses with a mortality rate of 50-90% in humans. Early detection of Ebola virus infection can save thousands of lives by enabling isolation of infected individuals from susceptible ones. Currently the available diagnoses can only detect Ebola infection after the onset of symptoms which typically takes place within 14-21 days of viral exposure. This time lag in diagnosis facilitates viral outbreak and is an obstacle for infection control efforts. Additionally, the available point of care (POC) diagnosis gives qualitative results, visually confirmed by two independent readers and in case of disagreement a third reader is required. Cold chain support is also required for their transport and storage which is often difficult to arrange in remote areas where the viral outbreaks occur. Therefore, the limitations of the current diagnosis costs valuable lives and calls for advancement in the method of diagnosis. Our goal is to develop a user-friendly and cost-effective portable device for reliable immediate detection of Ebola viruses at POC. For that purpose, we have chosen the Ebola virus soluble glycoprotein (sGP) as the biomarker, which is secreted in the host blood at a concentration of 137 ng/ml within 3 days of exposure. The innovation of our proposal lies in the combination of aptamer technology with up-converting phosphors (UCPs) to develop a lateral flow (LF) assay sensor. Aptamers are nucleic acids that bind their targets avidly and specifically and are selected in vitro by Systematic Evolution of Ligands by EXponential enrichment (SELEX). For biosensor development, aptamers have many advantages over antibodies, including their smaller size, ready and inexpensive synthesis, reliable production, amenability to chemical synthesis and modifications, adaptability to a broad range of assay format, and long-term stability at room temperature. Conjugation of aptamers with UCPs is also expected to lower background fluorescence signal, which will enhance the sensitivity of the proposed LF assay. In phase I, we will optimize the sensor resolution and sensitivity, deploying our developed DNA aptamers against sGP in human serum. If successful, our prototype will have an immense impact on the current diagnostic research by combining the robustness of aptamers with the sensitivity of UCPs and the operational ease of LF assays. Specifically, we will improve existing diagnostic method by offering: 1. Immediate detection: the presence of Ebola will be detected from the infected serum within 3 days of exposure in contrast to the current detection around 14-21 days of exposure. 2. Longer shelf life will be imparted without the need of cold chain support by the robustness of aptamers and 3: Low cost will also be imparted as the aptamers are more cost effective and more reliably produced compared to antibodies.