## 3 Results and Discussion
### SERS Measurements  
Figure 9 shows the SERS spectra of R6G solutions with concentrations of \(1\times 10^{-6}\) M, \(1\times 10^{-7}\) M, \(1\times 10^{-8}\) M, and \(1\times 10^{-9}\) M, respectively, using our SERS substrate with 10 oxidation-reduction cycles. The Raman band assignments of the main peaks for R6G were given in Figure 9. Even at a low concentration of 1 nM, characteristic peaks of R6G at 607, 765, 1176, 1304, 1355, 1503, and 1640 cm\({}^{-1}\) were clearly observed from these spectra. These spectra indicate that our SERS substrates have a good enhancement factor.  
Figure 8: A cross-sectional view of the electric field distribution between two micro-pyramids with an incident light wavelength of 633 nm. In this figure, the normalized electric field represents the ratio between the electric Field excited by the local surface plasmon resonance and the background electric field.  
We used the following well-established formula [21] to estimate the enhancement factor (EF) value of R6G:  
\[EF=(I_{SERS}/I_{bulk})/(N_{bulk}/N_{SERS}) \tag{1}\]  
where \(I_{SERS}\) is the vibrational intensity of the SERS of the R6G molecule at 1510 cm\({}^{-1}\), and \(I_{bulk}\) is the vibrational intensity of the normal Raman spectrum of the R6G molecule at 1510 cm\({}^{-1}\). \(N_{SERS}\) is the number of molecules irradiated by laser spots under SERS, and \(N_{bulk}\) is the number of molecules of the bulk sample irradiated by laser spots under normal Raman condition. The EF was calculated to be about \(1\times 10^{7}\), which is quite good for a gold-based SERS substrate [21; 22]. This result is assuming the SERS substrate is densely packed with R6G molecules after immersed in \(10^{-6}\) M R6G solution for 12 h. However, the R6G is likely to cover the substrate at a lower density. Therefore, the SERS enhancement factor calculated here is the lower limits.  
The developed SERS substrates were also tested with low concentration malachite green solution. Figure 10 shows measured SERS spectra for malachite green concentrations ranging from 0.01 nM to 10 nM on SERS substrates. The Raman characteristic peaks of malachite green at 1620 cm\({}^{-1}\), 1370 cm\({}^{-1}\), 1175 cm\({}^{-1}\), 916 cm\({}^{-1}\), and 802 cm\({}^{-1}\) are in-plane C-C stretching vibration of the ring, overlapping effect of in-plane N-phenyl stretching vibration and in-plane C-C stretching vibration, in-plane aromatic C-H vibration, ring-breathing, and out-of-plane phenyl-H bending, respectively. These characteristic peaks of malachite green are clearly observed from the spectra. According to the result, the SERS substrates were found to be able to detect a low concentration of 0.01 nM malachite green solution, which is very close to the finding in previous studies [23; 24]. The enhancement factors for different concentrations of malachite green were also calculated. The Raman characteristic peak values of malachite green at 1370 cm\({}^{-1}\) acquired from Figure 10 were used to calculate the enhancement factors for different concentrations of malachite green. The resulting enhancement factors for concentrations of 10 nM, 1 nM, 0.1 nM, and 0.01 nM malachite green are \(1.21\times 10^{7}\), \(1.49\times 10^{7}\), \(7.81\times 10^{7}\), and \(4.05\times 10^{8}\), respectively. It can be seen that when the concentration of the MG solution to be measured is lower than 1 nM, as the concentration decreases, the enhancement factor (or sensitivity) of the developed chip becomes larger, rather than a constant value.  
Figure 9: Shows the SERS spectra of R6G solutions with concentrations of \(1\times 10^{-6}\) M, \(1\times 10^{-7}\) M, \(1\times 10^{-8}\) M, and \(1\times 10^{-9}\) M, respectively, using our SERS substrate with 10 oxidation-reduction cycles.