Extraordinary SERS on nanostructured self-assembled silver films due to preliminary random nano-profiling of substrate and ultrasonic exposure of analyte during the deposition

In this study, we have investigated the surface-enhanced Raman scattering (SERS) spectra of myoglobin on silver substrates with different morphology. The aim was to determine the optimal parameters of analyte and substrate preparation for obtaining of high-amplitude SERS spectra of proteins. It is shown that not only the morphology of the silver film, but also the method of analyte molecules deposition on the SERS substrate plays an important role. Glass substrates were pretreated several stages in organic solvents before silver deposition. It is shown that pretreatment of the glass surfaces lead to a decrease in the silver SERS film roughness. Myoglobin was applied to the SERS substrates in the form of an aqueous solution and then it was precipitated under ultrasonic treatment at frequencies of 580 kHz and 47 kHz. SERS spectra were recorded at randomly selected locations of the dried precipitate using excitation wavelengths of 785 nm and 532 nm. Unusual form myoglobin SERS spectra were observed, in which only certain part of the spectrum demonstrated the additional enhancement in comparison to the overall spectrum. We called such spectra extraordinary ones. In the extraordinary myoglobin SERS spectra, the amplitude of the vibrational band at 996 cm⁻¹ is approximately five times more intense compared to other strong vibrational bands. And the ratio of amplitudes of vibrational band in extraordinary SESR spectra increased by more than an order of magnitude compared to the same ratio for amorphous myoglobin. The article discusses the mechanisms leading to the appearance of extraordinary SERS spectra.