## 3 Results
Another analysis was made on the same plates by comparing the results obtained from the substrates produced by varying the laser fluence, while maintaining a constant number of 50 overlapping pulses. In Figure 6, the spectra obtained for Cu-Au (b) and Cu (c) are presented. Apart from the strong signal of R6G on the Cu-Au substrate processed with 0.6 J/cm\({}^{2}\) fluence, for all the other fluences investigated there is no clear dependence of the SERS intensity on the laser fluence. The highest peak intensity was obtained for Cu at a fluence of 1.2 J/cm\({}^{2}\). For Cu-Au, a highly distinguishable intensity peak was obtained at 0.6 J/cm\({}^{2}\), as all the other fluences produced visibly lower amplification. There is a connection between the XPS intensity of Au (Figure 3) and the Raman signal intensity when varying the laser fluence (Figure 5), but the connection is nonexistent when varying the number of overlapping pulses in the case of Cu-Au substrates (Figure 6). Figure 5: Raman spectra for R6G with 10\({}^{-4}\) M concentration on Cu-Au, Cu and unprocessed Au substrates (**a**); surface-enhanced Raman Spectroscopy (SERS) efficiency for Cu-Au substrates (**b**) and Cu substrates (**c**) on the 1356 cm\({}^{-1}\) peak of R6G with 10\({}^{-4}\) M concentration, fabricated with different numbers of overlapping pulses and the same laser fluence of 1.2 J/cm\({}^{2}\); red lines (middle and right panels) are B-spline interpolations. Following this analysis, the best substrate was selected and its performance was compared with a commercial product for validation of the amplification results. The comparison product was chosen from SILMECO Gold SERS series [26,27], since the producers also use Au nanostructures for SERS. In this brief comparison, R6G spectral analysis was conducted and the result is presented in Figure 7--the highest intensity spectrum represents the commercial product SILMECO Au, the red line represents the best substrate from the present work (Cu-Au produced with 0.6 J/cm\({}^{2}\) laser fluence and 36 overlapping pulses) and the blue line represents the best Cu substrate (1.2 J/cm\({}^{2}\) and 36 overlapping pulses). The almost flat pink line represents an unprocessed Au substrate (thin film of Au on glass). Even though the same drop that contained R6G was also pipetted onto a thin film of Au, the lack of substrate processing led to insignificant amplification. By combining Cu and Au in a biplasmonic substrate, a superior signal amplification is observed, compared with the simple Cu substrate. The Raman signal is sufficiently amplified by the SERS effect given by the use of Cu-Au substrate, providing that the Raman signal intensities are just a few times lower than a commercial substrate. Figure 6: Raman spectra for R6G with 10\({}^{-4}\) M concentration on Cu-Au substrates (**a**); SERS efficiency for Cu-Au substrates (**b**)and Cu substrates (**c**) on the 1356 cm\({}^{-1}\) peak of R6G with 10\({}^{-4}\) M concentration, fabricated with different laser fluences and fixed 50 overlapping pulses; red lines (middle and right panels) are B-spline interpolations. Figure 7: Comparison of Raman spectra for R6G with 10\({}^{-4}\) M concentration on SILMECO-Au, Cu-Au (200 nm) laser ablated, Cu laser ablated and a flat Au substrates acquired in the same conditions: (**a**) raw spectra; (**b**) fluorescence corrected spectra.