1a.Objectives (from AD-416):
The long term goal of this project is the development of rapid, reliable, and sensitive diagnostic assays for the identification of zoonotic viruses that are suitable for use in point-of-care facilities, as well as in the field for use during times of natural or intentionally caused disease epidemics.
1b.Approach (from AD-416):
The feasibility of this goal will be demonstrated in the proposed research by the development of Au nanoparticle-mediated indirect and direct capture assays for arboviruses including Rift Valley Fever Virus (RVFV) or antibodies to these viruses. The detection technology is based upon surface enhanced Raman scattering (SERS) spectroscopy, an analytical tool that is well known for its high detection specificity and sensitivity and which affords several advantages over other nucleic acid detection and immunological technologies currently in use. This includes the reduction in assay costs by affording sensitivity without current amplification technologies and easy adaptation to point-of-care use. The primary focus of the proposed studies will be the demonstration and evaluation of the relative efficacies of alternative in vitro model assays, followed by the translation of these findings to the development of an optimized assay for detecting genetic material, antigen or antibodies to arboviruses.
Efforts to develop SERS as a next-generation analyte detection modality for clinical and point of care test applications have focused on two priority research objectives in the past year. The first, which will hopefully improve upon the high-level detection sensitivities we previously reported for viral antigens using gold nanoparticles (GNPs) and paramagnetic nanoparticles (PMPs), is the development of a single nanoparticle assay. The use of combined functionality nanoparticles affords a reduction in the complexity of reagent fabrication procedures and the opportunity for an increase in detection sensitivity attributed to antigen capture using only a single particle as opposed to the requirement of antigen co-recognition using the two-particle GNP/PMP system. Currently, we have successfully synthesized Au-coated PMPs of reproducible and defined size, and shown that they can be used to support SERS enhancements that are equivalent, if not superior, to enhancements that are recorded using commercially supplied GNPs. Experiments underway include the fabrication of Au-coated PMPs with antibodies recognizing arbovirus antigens and the optimization of the performance attributes of SERS immunoassays using these particle reagents. We have also initiated studies to determine whether Au-coated PMPs can be used to sensitively detect micro RNAs that correlate with different mammalian disease conditions.
The other research area to which we’ve devoted considerable effort has been the optimization of SERS-based magnetic capture immunoassays. These studies have entailed a determination of the conditions needed to provide minimum assay reaction times (i.e. < 10 min.), the rigorous evaluation of particle sizes and concentrations required for maximum detection sensitivity, and the development of antibody-functionalized particles optimized for surface coverage levels and capture probe orientation. We have recorded improvements in detection sensitivities using FAb fragments prepared from anti-viral IgGs and are now embarking on studies using single chain variable fragments consisting of recombinant fusions of the light and heavy variable chains of antibody to the envelope protein of West Nile virus.