## 4 Conclusions  
It is shown that the amplitude of Raman spectra of an air-dried protein solution strongly depends on the parameters of the surface morphology at the nanoscale level. In this paper, we demonstrated low concentration myoglobin detection down to 10 ug/mL using Raman amplified signal (at the excitation wavelength of 785 nm) from SERS-active substrates with e-beam evaporated self-assembled 100 nm-thick silver thin films on the surface of silicon (111), glass and mica substrates. We observed up to \(10^{5}\) times amplification (sample type IV) of the spectrum compared to 100 nm-thick aluminum thin film control sample. We demonstrated that Raman signal amplification depends dramatically on the silver SERS-active substrates surface morphology. The characteristic dimensions of surface morphology elements of considered SERS substrates were in the range from one to tens of nanometers. Using optical confocal microscopy, it was shown that the protein forms a conformation on the surface of a self-assembled silver film with a specific morphology, which makes it possible to effectively implement efficient Raman signal amplification. The most significant surface morphology parameters the self-assembled silver films and corresponding deposition recipe parameters to obtain high-amplitude Raman spectra were identified. We suggested the set of surface morphology parameters (Rq, R3z, Rku, Wq, \(\lambda\)a) in combination with the Raman spectra and deposition recipe (deposition rate, substrate temperature, vacuum conditions) parameters, which have to be taken into account for high-quality SERS-active substrates nanoengineering. In order to recover valuable data in reasonable time with such a huge number of parameters, which have to be optimized, we suggested using machine learning methods. Finally, we utilized this approach for quick and precise automatic analysis of a vast number of fabricated SERS substrates, which made it possible to find optimal deposition recipe parameters for SERS-active substrates nanoengineering. We found out that to provide the highest Raman signal amplification, one have to fabricate SERS-active substrates with the following surface morphology: root-mean-square surface roughness (Rq) equal to 5 nm, the height difference of the third largest peak to the third largest depression (R3z) equal to 13.2 nm, kurtosis (Rku) equal to 16.5, mean-square waviness (Wq) equal to 57.5 nm, average wavelength of the profile (\(\lambda\)a) equal to 590 nm. Based on these requirements  
Figure 12: Distribution of substrate new parameters for (**a**) Neuron1, (**b**) Neuron3, (**c**) Neuron 6. Number of sites at ordinate. Neural values at abscissa. we developed the SERS-active substrates fabrication process based on self-assembled continuous silver films, which provides reproducible Raman signal amplification up to \(10^{5}\) times compared with 100 nm-thick aluminum thin film control sample. It is worth noticing that proposed SERS-active substrates require no additional expensive technological operations (like e-beam lithography, dry etching, etc. ), promote high-quality reproducible probe preparation using cheap in-air drying technique and provide much higher thermal conductivity compared to island-like SERS substrates leading to much higher possible excitation laser power (up to 65 mW). The metal thin films growth process was developed by [PERSON] and [PERSON]. [PERSON], [PERSON] and [PERSON] performed the AFM and SERS measurements and writing-original draft preparation. [PERSON] developed data mathematical treatment algorithms and performed data treatment. [PERSON] and [PERSON] analyzed the data, participated in measurements, review and editing the manuscript [PERSON]. ([PERSON]) and [PERSON] ([PERSON]) planned all the experiments, participated in technology development, writing--review and editing the manuscript and analyzed the data. [PERSON] supervised the study and funding acquisition. All authors contributed to writing the manuscript.