## 4 Discussion  
In the present work, the potential of LoC-SERS as a spectroscopic based method was demonstrated for the simultaneous detection of PZA and POA in a microfluidic device. The significant differences in the SERS spectra of PZA and POA were determined, e.g., for PZA specific marker bands are centered at 1218 \(\upmu\)m\({}^{-1}\) and 718 \(\upmu\)m\({}^{-1}\), for POA the marker bands are centered at 731 \(\upmu\)m\({}^{-1}\) and 852 \(\upmu\)m\({}^{-1}\). The flow rates in the microfluidic device could be optimized to achieve best signal to noise ratio when citrate (concentration 10\({}^{-5}\) m) was added (5 nL/s) and the flowrates of mineral oil, AgNO\({}_{3}\)/NH\({}_{3}\) solution and N\({}_{2}\)H\({}_{4}\) was set to be 30 nL/s, while analyte flowrate was set to 10 nL/s.  
Figure 5: (**a**) LoC-SERS mean spectra of mixtures of PZA (gray) and POA (black) in water (using y-AgNPs). Mixing ratios of analyte stock solutions (10\({}^{-4}\) M) are indicated in the graph. Mixing was realized before injection into the chip. The standard deviation values are indicated in light gray; (**b**) peak intensities of the marker bands at 1218 \(\upmu\)m\({}^{-1}\) (for PZA) and 852 \(\upmu\)m\({}^{-1}\) (for POA) relative to the peak at 1472 \(\upmu\)m\({}^{-1}\), respectively.  
In aqueous conditions for both analytes the SERS signal vs. concentration yields a sigmoidal curve. However, the limit of detection was calculated to be 27 \(\upmu\)M for PZA and 21 \(\upmu\)M for POA when detected discretely each one. Thus, it could be shown as a proof of principle that the discrimination between PZA and its metabolite POA can be realized with a very high sensitivity. Furthermore, the two analytes were simultaneously detected in aqueous solution with varying concentrations, it was demonstrated that a linear dependency of the Raman signal with respect to the concentration holds true in the range of 20 \(\upmu\)M to 100 \(\upmu\)M for PZA and 50 \(\upmu\)M to 100 \(\upmu\)M for POA, respectively. The detection limits for PZA and POA in a mixture of both analytes are 20 \(\upmu\)M and 50 \(\upmu\)M, respectively. The ratio of the marker modes of PZA and POA can be a relevant indicator for PZA resistant MTB.  
The following are available online at [[http://www.mdpi.com/2076-3417/9/12/2511/s1](http://www.mdpi.com/2076-3417/9/12/2511/s1)]([http://www.mdpi.com/2076-3417/9/12/2511/s1](http://www.mdpi.com/2076-3417/9/12/2511/s1)), Figure S1: Droplet based microfluidic chip, Figure S2: Determination for the limit of detection for PZA and POA solved in high purity water, Table S1: band assignment for PZA and POA according to the cited literature, Measurement overview.  
Conceptualization, I.J.J., P.S., M.Z. and D.C.-M.; Formal analysis, A.M., M.J.; Funding acquisition, M.Z. and D.C.-M.; Investigation, J.H. and P.S.; Methodology, A.M.; Project administration, K.W. and D.C.-M.; Supervision, J.P.; Writing--original draft, A.M. and I.J.J.; Writing--review & editing, K.W., P.S., M.Z., D.C.-M. and J.P.  
We gratefully acknowledge the Federal Ministry of Education and Research, Germany (BMBF) for supporting the InfectConics (13 GW0096F) and EXASENS (13N13856) project grants as well as BMBF and German Aerospace Center (DLR) for supporting the Myco-NET2 (01 DN15028) project grant. We thank for funding the projects ERANET LAC EALCA2014/HID-0352, CONCYTEC-FONDECYT-Peru 086-2015, and the Wellcome Trust Intermediate Fellowship (grant 099805/Z/12/Z).  
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.