Bi-analyte demonstration of non-resonant, single-molecule SERS with isolated lithographic enhancement structures.

Single molecule Raman spectroscopy using isolated, circular ~ 110 nm diameter, multilayer disk enhancement structures fabricated with 355 nm interferometric lithography in a 2D 400 nm period array is reported. Adenine and cytosine are selected as a bi-analyte pair, with comparable strength Raman lines arising from ring breathing modes in proximity (A: 743 cm ^-1 shift and C: 807 cm ^-1 shift). Samples are soaked in 1:2 A:C mixtures diluted in water to ~ 1x10 ^-3 to 5x10 ^-4 of a monolayer coverage. Raman spectra are obtained with 633 nm excitation. The spectra show Poisson statistics with individual spectra showing: only A; only C; and A & C features demonstrating single molecule sensitivity. Over 90% of the spectra are null spectra without either A or C features. Importantly, the enhancement structures are isolated from each other, no close approach is required as in the case of spherical nanoparticles where ~ 1 nm gap modes are responsible for the large enhancement required for non-resonant single molecule sensitivity.