By taking advantage of the spectral properties of metal carbonyls, we have designed a surface-enhanced Raman spectroscopy (SERS) ratiometric assay for measuring cell-free circulating DNA (cfDNA) from Epstein-Barr virus in blood for nasopharyngeal carcinoma (NPC). This assay consists of a rhenium carbonyl (Re-CO) to serve as a DNA probe, an osmium carbonyl (Os-CO) embedded within the SERS-active substrate as an internal reference, and a streptavidin layer on the surface of the substrate. Hybridization of cfDNA with biotinylated-capture sequence leads to immobilization of cfDNA on the substrate. The binding of Re-CO via daunorubicin (DNR) to cfDNA is accompanied by an appearance of a strong symmetry stretching vibrations peak at 2113 cm, which has spectral overlap with Os-CO (2025 cm). This results in an increase in the I/ I ratio and quantitatively correlates with cfDNA. This SERS assay can be readily used to detect cfDNA in blood samples from patients due to the intensity ratio of I/ I lying in a silent region (1780-2200 cm) in the SERS spectrum of the biomolecules.
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