## 3 Results  
### PZA and POA: Spectral Characterization  
Raman spectroscopy is known to provide highly specific signals and to allow the discrimination of molecules based on the achieved information in the vibrational spectra. POA is the active metabolite of PZA, its absence or presence in a very low amount in in vitro MT cell cultures after the administration of PZA might indicate PZA resistance of the bacteria culture. Therefore, it is essential to implement an analytical method for the analysis and accurate detection of POA alone or POA/PZA mixtures and discriminate this from samples containing just the prodrug PZA. The two targeted substances are structurally very similar molecules (Figure 1). Both share a pyrazine ring, whereas PZA has an amide moiety and POA a carboxylic acid group. This is why, the reference Raman spectra measured on the pure powder (Figure 1a,b; gray spectra at the bottom) show besides some common bands also several modes specific for each molecule. The detailed assignment of the Raman bands has been already reported in the literature [37; 38; 40; 41]. The marker bands for the pyrazine ring are the doublet present in the spectrum of both molecules at 1021 and 1055 cm\({}^{-1}\) corresponding to the in-plane bending of the pyrazine ring and the CN stretching mode of the same ring at 1528 cm\({}^{-1}\). In the high wavenumber range the mode of the CH stretching vibrations arises at 3066 cm\({}^{-1}\). Specific marker bands for PZA are the band at 1080 cm\({}^{-1}\) (rocking of the NH\({}_{2}\) group) and the band at 1440 cm\({}^{-1}\) (bending of the NH\({}_{2}\) group). The significant marker band for POA is located around 1710 cm\({}^{-1}\), ascribed to the C=O stretching of the carboxylic moiety. Although Raman spectroscopy provides a very high specificity, the sensitivity that is required for the discrimination of PZA susceptible and PZA resistant MTB can only be achieved by enhancing  
Figure 1: Reference Raman spectra of the powder (gray spectra in the bottom) and surface-enhanced Raman spectroscopy (SERS) spectra (black spectra in the top), applying hydroxyl amine hydrochloride reduced h-AgNPs, of PZA and POA in a concentration of \(10^{-3}\) M in panel (**a**) and (**b**), respectively. Marker bands for both analytes are indicated with black lines, marker band specific for PZA are depicted with doted lines, marker band for POA with dashed lines. For both analytes a scheme of the molecular structure is shown. the Raman signal. However, exploiting the plasmon-based surface enhancement effect, the presence of plasmonic NPs can significantly change the spectral profile of the SERS spectra as compared to the Raman spectra. This is associated with surface dependency effects, e.g., orientation to the surface (surface selection rules), gradient field effect and charge-transfer events, which results in variations of the spectral position and/or intensity of certain peaks [51; 52]. In the SERS spectrum of PZA and POA (aqueous solution, concentration \(10^{-3}\) M, h-AgNP) measured with hydroxyl amine hydrochloride reduced batch silver nanoparticles h-AgNPs ((Figure 1a,b) top panel) the three Raman modes characteristic for the fundamental vibrations of the pyrazine ring (1021 cm\({}^{-1}\), 1055 cm\({}^{-1}\) and 1528 cm\({}^{-1}\)) are still clearly visible and unaffected by the proximity of the silver surface. For PZA, a new band caused by the symmetric stretching of the amide group of the pyrazine ring appears in the SERS spectra at 1218 cm\({}^{-1}\)[53]. Furthermore, the signal centered at 718 cm\({}^{-1}\) in the SERS spectra is assigned to the OCN bending vibration. Considering the observed changes in the SERS-spectra as compared to the Raman spectra and the surface selection rules it is possible to assume the orientation of the PZA molecules relative to the surface. Most likely the interaction takes place through the nitrogen atom, resulting in the upright position of the aromatic ring structure relative to the metal surface.