RESUMO
An indirect capture model assay using colloidal Au nanoparticles is demonstrated for surface enhanced Raman scattering (SERS) spectroscopy detection of DNA. The sequence targeted for capture was derived from the West Nile Virus (WNV) RNA genome and selected on the basis of exhibiting minimal secondary structure formation. Upon incubation with colloidal Au, hybridization complexes containing the WNV target sequence, a complementary capture oligonucleotide conjugated to a strong tethering group and a complementary reporter oligonucleotide conjugated to methylene blue (MB), a Raman label, anchors the resultant ternary complex to Au nanoparticles and positions MB within the required sensing distance for SERS enhancement. The subsequent elicitation of surface enhanced plasmon resonance by laser excitation provides a spectral peak signature profile that is capture-specific and characteristic of the Raman spectrum for MB. Detection sensitivity is in the submicromolar range and was shown to be highest for thiol, and less so for amino, modifications at the 5' terminus of the capture oligonucleotide. Finally, using Quartz Crystal Microbalance-Dissipation as a tool for modeling ternary complex binding to Au surfaces, quantitative measurements of surface mass coverage on Au plated sensor crystals established a positive correlation between levels of ternary complex adsorption and their correspondent levels of SERS signal intensification. Adapted to a compact Raman spectrometer, which is designed for analyte detection in capillary tubes, this assay provides a rapid, mobile and cost effective alternative to expensive spectroscopic instrumentation, which is often restricted to analytical laboratories.
