SERS molecular sentinel for the RNA genetic marker of PB1-F2 protein in highly pathogenic avian influenza (HPAI) virus.

We have developed a simple and sensitive assay for the detection of the RNA genetic marker associated with high pathogenicity influenza (HPAI) virus. The assay constituted of an array of Raman label tagged hairpin-DNA immobilized on a surface-enhanced Raman scattering (SERS) active substrate as the molecular sentinel (MS) reporter. Upon incubation of the assay with the target RNA, the structure of the hairpin-DNA probe changed from stem-loop configuration (closed state) to DNA/RNA hybridization configuration (open state) so that the Raman label tag will be physically separated from the SERS substrate and induce a decrease of Raman scattering intensity. A metal film over nanosphere (MFON) substrate was developed with a SERS enhancement of about 1.7 × 10(5). Based on this MS-modified substrate, the SERS signal showed a linear relationship to the target RNA in the range of 0-60 attomoles and the limit of detect is 2.67 attomoles. The non-complementary RNA sequences control was also detected and no spectral response was observed. The sensing process only required a single hybridization step and post-hybridization washing could also be omitted. Given that this ultrasensitive biosensor assay is free of polymerase chain reaction (PCR) amplification, it would be a potential diagnostic tool for point-of-care HPAI virus detection.