Detection of genetic markers related to high pathogenicity in influenza by SERS

We have developed a method for the detection of genetic markers associated with high pathogenicity in influenza. The assay consists of an array of 5'-thiolated ssDNA oligonucleotides immobilized on the surface of a Ag nanorod substrate that serve as capture probes for the detection of synthetic RNA sequences coding for a genetic mutation in the influenza PB1-F2 protein. Hybridization of the DNA probes to their complementary RNA sequences was detected using surface-enhanced Raman spectroscopy (SERS). Multivariate statistical analysis was used to differentiate the spectra of the complementary DNA probe-RNA target hybrids from those of the non-complementary DNA probes containing a single base pair polymorphism. Hierarchical cluster analysis (HCA) was able to distinguish with 100% accuracy the spectra of the complementary DNA probe-RNA target from the spectra of the immobilized DNA probes alone, or the DNA probes incubated with non-complementary RNA sequences. Linearity of response and limits of sensitivity of the SERS-based assays were determined using a partial least squares (PLS) regression model; detection limits computed by PLS was determined to be ~10 nM. The binding affinity of the DNA probes to their complementary RNA sequences was confirmed using enzyme-linked immunosorbent assay (ELISA); however, the detection limits observed using ELISA were approximately 10× higher (~100 nM) than those determined by PLS analysis of the SERS spectra.