## 3 Results
### Simultaneous Detection of PZA and POA in Aqueous Solution  
To prove the high potential of the LoC-SERS device for providing reliable data for the PZA resistance discrimination, first PZA and POA were detected simultaneous in an aqueous solution. In the supernatant of the PZA treated mycobacteria only PZA might be found in the case of PZA resistance. However, if the bacteria are PZA susceptible a mixture of PZA and POA will be found in the supernatant. Therefore, depending on the conversion rate of PZA to POA, the time of analysis and presence of PZA resistance, POA might be present in a mixture with PZA or might be completely absent. Consequently, it is of interest to discriminate between samples containing only PZA, a mixture  
Figure 4: (**a**) LoC-SERS mean spectra of different concentrated POA in water as indicated in the plot. The standard deviation values are indicated in grey; (**b**) the integrated intensity of the marked peak centered at 852 cm\({}^{-1}\).  
Figure 3: (**a**) Lab-on-a-chip SERS (LoC-SERS) mean spectra of different concentrated PZA in water as indicated in the plot. The standard deviation values are indicated in grey; (**b**) the integrated intensity of the marked peak centered at 1218 cm\({}^{-1}\).  
of PZA and POA and only POA. As a proof of principle, samples with different prodrug-metabolite ratios were measured. As discussed above, the two molecules show, besides common bands ascribed to the vibrations of the pyrazine ring, also vibrational modes characteristic for the different analytes. It is expected that these differences in the SERS spectra will also be visible in a mixture. Seven different mixtures were measured using the LoC-SERS setup, the concentration of PZA ranging from 0 to 100 \(\upmu\)M and the concentration of POA from 100 to 0 \(\upmu\)M. The means of the measured SERS spectra are depicted in Figure 5a. Differences are obvious in band intensity ratios, positions and the presence/absence of the specific marker bands.  
The mayor differences can be noticed in bands located at 852 and 1372 \(\upmu\)m\({}^{-1}\) characteristic for POA and the vibrational modes centered at 1218 and 1440 \(\upmu\)m\({}^{-1}\), being marker modes for PZA. These bands are changing in ratio correlated to the individual concentrations of both target molecules. In Figure 5b, the peak intensity of the PZA marker mode at 1218 \(\upmu\)m\({}^{-1}\) and the POA marker mode at 852 \(\upmu\)m\({}^{-1}\) are plotted after normalization to the peak at 1472 \(\upmu\)m\({}^{-1}\). The reference band centered at 1472 \(\upmu\)m\({}^{-1}\) is caused by N-C stretching [38]. This band is present in the spectra of both analytes (as indicated in Figure 1a,b) with a resembling intensity. Thus, this band is very well suited as a reference band for the normalization. For both analytes a linear signal to concentration dependency could be observed. For PZA the concentration ranged from 20 to 100 \(\upmu\)M (with a coefficient of correlation R2 = 0.99) and for POA the range from 50 to 100 \(\upmu\)M (with a coefficient of correlation R2 = 0.92) could be determined. Thus, for the concentration PZA/POA 20 \(\upmu\)M/80 \(\upmu\)M to 50 \(\upmu\)M/50 \(\upmu\)M to simultaneous detection could be realized as very successful. The detection limits for PZA and POA are 20 \(\upmu\)M and 50 \(\upmu\)M, respectively.