SERS Nanoprobe for Intracellular Monitoring of Viral Mutations

Gold nanostars, functionalized with thiolated DNA hairpins bearing a Raman-active fluorescent dye at the 3' terminus, were engineered to identify and quantify RNA mutations in the influenza A virus (IAV) genome employing surface enhanced Raman spectroscopy (SERS). The DNA hairpin structure was designed to selectively extend/fold in the absence/presence of the viral RNA targets, resulting in the fluorophore being brought away from or close to the gold nanostar surface, leading to an OFF-ON switching of the SERS signal. Validation of the switchable SERS nanostar probes was first carried out in buffer, showing that the detection is sequence-specific and that the high sensitivity provided by these SERS probes allows target detection at the single particle level. We also demonstrate that the degree of signal recovery can be closely correlated with the number of genetic mutations. Further experiments carried out with HeLa cell lysate spiked with RNA oligonucleotides demonstrate that the functionality of these nanoprobes were not detrimentally affected by the complex matrix. As a proof of concept, we also tested these nanoparticle probes in vitro by specifically targeting the hemagglutinin (HA) segment in live HeLa cells transfected with plasmids coding for either HA or two other IAV segments, PB1 and PB2, as negative controls. The intracellular SERS response in individual transfected HeLa cells demonstrates high sequence-selectivity of the probes for the HA segment, suggesting the applicability of these probes for multiplexed detection and quantification of viral RNAs in individual cells with an approach that can account for the viral population diversity. This also represents the first time that molecular beacon-based SERS probes have been employed to detect viral RNA target in intact individual cells.