## 2 Materials and Methods  
Silver thin films were deposited on prime-grade intrinsic Si(111) FZ high-resistivity wafers (>10,000 \(\Omega\)-cm), glass and muscovite mica substrates using both a 10 kW e-beam evaporator (Angstrom Engineering, Canada) with a base pressure lower than \(3\times 10^{-8}\) Torr and 8 kW e-beam evaporator (Quartz, Russia) with a base pressure lower than \(5\times 10^{-6}\)Torr. Before loading into the vacuum chamber of an electron-beam evaporator the substrates were preliminary cleaned with isopropanol (99.5%) (Sigma-Aldrich, USA). Mica substrates were cleaved perpendicular to the c-axis to reveal fresh surfaces, prior to deposition. Prepared substrates were placed in a substrate holder at a distance of 380 mm from the source of metal vapor. Before the metal film deposition, the substrates were pre-cleaned in plasma in a residual atmosphere at a pressure of \(10^{-3}\)Torr in the vacuum chamber. All the films were grown using high-purity 4N (\(\geq\)99.99%) (Moscow special alloys processing plant, Russia) granulated silver with the grain size of 3 mm. Seven types of SERS substrates were obtained and the possibility of myoglobin SERS-based detection (in a solution with concentrations of 10 ug/mL) was further studied. Type I: Si (111)/Ag single-crystalline (65 nm). Type II: Si (111)/Ag single-crystalline (65 nm)/Al\({}_{2}\)O\({}_{3}\) (atomic layer deposition (ALD)) (2 nm). Type III: glass/Ag rough film (100 nm). Type IV: glass/Ag rough film (100 nm) with \"droplet phase\". Type V: mica/Ag single-crystalline film (100 nm) with pore-like semi-regular nanostructures. Type VI: mica/Ag single-crystalline film (100 nm). Type VII: glass/Al film (3000 nm)/Ag rough film (100 nm). The samples of types I, II, V, VI were made at the BMSTU Nanofabrication Facility (Functional Micro/Nanosystems, FMNS REC, ID 74300). The samples of types III, IV, VII were made at the ITAE RAS. During films evaporation the deposition rate and the substrate temperature were varied, which made it possible to control the microstructure of growing films at the nanoscale level. The chamber pressure for the samples Type III, Type IV, Type VII was \(5\times 10^{-6}\)Torr, cathode voltage was 12 V, beam current was 30 mA, source accelerating voltage was 8 kV. For the samples Type I, Type II, Type V, Type VI, the chamber pressure was \(3\times 10^{-8}\) Torr. The deposition rate varied from 1 nm/s to 30 nm/s, the substrate temperature ranged from 300 K to 650 K. The thickness of the films during deposition was controlled directly in the process of growth by means of optical control. After deposition, the film thickness was selectively measured using a stylus profilometer KLA-Tencor P-17 (KLA-Tencor Corporation, USA). Surface morphology was investigated using a Solver-Pro atomic-force microscope of (NT-MDT, Russia). Images were taken in the tapping mode. The HA_NC/Au probes with resonance frequency 140 kHz \(\pm\) 10%, force constant 3.5 N/m, tip radius less than 10 nm were used. We used freshly prepared solutions of myoglobin (produced by Sigma-Aldrich) in deionized water with a concentration of 10 ug/mL. To determine the influence of topology and volumeof protein precipitate on the amplitude of SERS spectra, myoglobin solutions of high concentration (about 1 g/L)were used. Deionized water for the solution was produced using the Milli-Q purification system. Drops of a 3 \(\upmu\)L protein solution were applied on the surface of a silver film using a high-precision Eppendorf variable-volume pipettes. Drying was carried out on air under normal conditions. After drying, the Raman spectra of protein on the substrates were obtained using the Raman spectrometer (WITec, Germany) at the excitation wavelength of 785 nm. Laser power was 65 mW, spectrum accumulation time was 60 s. The lens 50X/0,8 L 0/\(\infty\) with chromatic aberration correction was used to record the spectra.