Quantifying RNA mutations at the single cell is critical for detection of viral mutations and ultimately for viral population control, especially in highly mutating viruses such as the influenza virus1. To that end, creating a highly sensitive tool that can track viral mutations intracellularly will provide mechanistic insight into viral evolution. We are designing a nanoparticle platform consisting of gold nanostars with a switchable surface enhanced Raman spectroscopy (SERS) - fluorescent beacons that allows for accurate and high resolution single particle-based influenza A virus (IAV) population sequencing without the need of PCR amplification. The nanoparticle platform has been developed to accommodate multiplexed intracellular imaging and sensing of various IAV gene sequences to account for viral population diversity2. The switchable SERS-fluorescent nanostar probes are designed to become active in the presence of their viral RNA targets with signal intensity varying with the number of mutations present on the target. Additionally, the nanostars are synthesized to provide optimal Raman scattering by enabling surface conjugation in proximity to hot spots3. Upon intracellular uptake, the particles are monitored via fluorescence in the absence of target (OFF state) and will provide quantitative detection of viral RNA by the use of SERS ratios and calibration curves (ON state). The switchable nanoparticle probe is a promising method for intracellular imaging and quantification of IAV genome that can potentially be expanded to other types of viruses as well.
1.Cella, L. N., Blackstock, D., Yates, M. A., Mulchandani, A. & Chen, W. Detection of RNA viruses: current technologies and future perspectives. Critical reviews in eukaryotic gene expression 23, 125-137 (2013).
2. Hutchinson, E. C., von Kirchbach, J. C., Gog, J. R. & Digard, P. Genome packaging in influenza A virus. The Journal of general virology 91, 313-328, doi:10.1099/vir.0.017608-0 (2010).
3. Indrasekara, A. S. et al. Gold nanostar substrates for SERS-based chemical sensing in the femtomolar regime. Nanoscale 6, 8891-8899, doi:10.1039/c4nr02513j (2014)