## 3 Results and Discussion
### Myoglobin Raman Spectra Measurements on the SERS-Active Substrates  
The SERS signal amplification of the proposed SERS-active substrates was evaluated using control sample and the protein deposited on the surface with the same drying technique. We have chosen the substrate with the aluminum thin film (100 nm), evaporated using the same PVD tool, as the control substrates. The control sample was the amorphous mass with the analyte concentration on the outer circumference of the drop in the form of a well-known \"coffee-ring\". Reproducible spectra of myoglobin, equally distributed over the spot boundary region, was obtained. It was found thickness of protein precipitate exceeded the spectral volume of our confocal microscope. The spectrum of myoglobin on a control sample with the 100 nm thick aluminum film is shown in Figure 2. The presented spectrum is typically interpreted for myoglobin according to [37; 48]: bands at 847 cm\({}^{-1}\) - Trp; 930 cm\({}^{-1}\) - \(\alpha\)-helix; 1004 cm\({}^{-1}\) - Phe; 1055 cm\({}^{-1}\) - Tyr; 1445 cm\({}^{-1}\) - Amide III; 1632 cm\({}^{-1}\) - Amide I; 1380 cm\({}^{-1}\) - Heme; 1415 cm\({}^{-1}\) - Trp. Some bands of oscillations are not developed, which may be due to the low signal-to-noise ratio. The analysis of dried protein substance on the surface of all the SERS substrates showed that the protein formed reproducible amorphous structures (Figure 3a). The SERS spectra were obtained from myoglobin on all the types of substrates except type VII. Silver films does not have their own Raman spectrum. In all the cases, the protein spectra were reproducible and did not depend on the measurement point. Therefore, in Figure 3b we present one typical protein spectrum with a typical amplitude. The location of the spectrum measurement is shown as the cross marker. The lines in the spectrum for ordinary air drying are decoded as follows according to the literature [37; 48; 49; 50]. The band at 362 cm\({}^{-1}\) is Fe-S vibrations; 418, 525 cm\({}^{-1}\) is S-S vibration; 603, 627 cm\({}^{-1}\) is C-S vibrations; 770 cm\({}^{-1}\) is Trp; 856, 892 cm\({}^{-1}\) is Tyr; 927 cm\({}^{-1}\) is \(\alpha\)-helix; 1052 cm\({}^{-1}\) is Phe; 1131 cm\({}^{-1}\) is v (C - N);1252 cm\({}^{-1}\) is Amide III; 1229 cm\({}^{-1}\) is \(\alpha\)-helix, Amide III; 1352, 1391 cm\({}^{-1}\) is Heme; 1441, 1467, 1607 cm\({}^{-1}\) is 8 (CH\({}_{\rm x}\)). We observed slight shifts in vibration bands, for example, at 1052 cm\({}^{-1}\) instead of 1004 cm\({}^{-1}\) which are associated with the conformation of a protein molecule while it interacts with the surface of silver films after drying, since the degrees of freedom of the molecule change as a result of electrostatic forces in solution. The phenylalanine band position was calculated theoretically earlier [46], in our experiments we observed the existence of three possible bands, which could be an indication of its oscillation. The band at 1004 cm\({}^{-1}\) is most often manifested in the spectra, but in our case it gives the band at 1052 cm\({}^{-1}\). Comparative analysis of the spectra lines amplitudes showed that the effective amplitude of the obtained Raman signal was increased relative to the control sample in all the cases, but for the best sample up to 10\({}^{5}\) times amplification was observed. A possible amplification mechanism could be described by theoretically predicted amplification values for rough films with sinusoidal roughness [35]. From the other hand, it could the result of the local field enhancement [19] in borehole of pore-like surface structures (see Figure 1f,g). The size of such pores is much smaller than the spectral volume of the microscope. We believe, that we experimentally observed the effective composite amplification as a result of combined action of these two mechanisms. Table 1 summarized the characteristic intensities of the myoglobin Raman signal for all the types of SERS substrates.