Published: September 14, 2011 r2011 American Chemical Society 8337 dx.doi.org/10.1021/ac2015799 | Anal. Chem. 2011, 83, 8337–8340 TECHNICAL NOTE pubs.acs.org/ac Online-Calibration for Reliable and Robust Lab-on-a-Chip Surface Enhanced Raman Spectroscopy Measurement in a Liquid/Liquid Segmented Flow Anne M€ arz, † Thomas Bocklitz, † and J€ urgen Popp* ,†,‡ † Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller University Jena, Helmholtzweg 4, 07743 Jena, Germany ‡ Institute of Photonic Technology (IPHT), Albert Einstein Straße 9, 07745 Jena, Germany b S Supporting Information T he interest of surface enhanced Raman spectroscopy (SERS) utilized in microfluidic lab-on-a-chip (LOC) devices is constantly growing in the field of bioanalytics 1 and medical diagnosis. 2 As the combination of SERS and a microfluidic device provides a sensitive and highly specific analytical tool, which allows handling of small sample volumes and low analyte concentrations, 3,4 LOC-SERS is well suited for such application fields. However, for the analysis of biomolecules by means of Raman or surface enhanced Raman spectroscopy, reliable data- bases and statistical data evaluation procedures are required. 5 To achieve these requests for investigation and identification of analyte molecules by Raman or surface enhanced Raman spec- troscopy, a sufficient number of spectra for the databases as well as a high comparability to the respective analyte spectra has to be provided. Establishing a database with an adequate amount of spectra for LOC-SERS experiments is fairly easy to accomplish. 6 Unfortunately, the reproducibility and comparability of the LOC-SERS spectra can be negatively impacted by the applied measurement procedure and inconsistent ambient conditions. For the LOC-SERS measurement principle, it has been shown that the application of a so-called segmented flow tremendously increases the reproducibility of the SERS spectra compared to measurements in continuous flow. 4 The fluctuating ambient parameters however appear to become a major issue. Already small variations in environmental parameters like, e.g., small fluctuations in temperature during the measurement, lead to an incomparability of the spectra due to wavelength shifts of the excitation laser line. This results in an error as a Raman- and SERS-spectra are measured relative to a fixed excitation wave- length (532.11 nm) and hence bands virtually are shifted, if the true excitation wavelength is altered. Subsequently, this becomes a crucial problem concerning the achievement of a reliable statistical analysis. The resulting statistical models will be unstable and inapplicable for real-world applications. This is an essential concern especially with regard to LOC-SERS as the method for point-of-care applications or transportable setups since these kind of analytical tools implicate rather inconsistent ambient conditions. In this contribution, we will demonstrate the influence of inconsistent environmental parameters on LOC- SERS measurements due to the impact on the setup and provide a solution negotiating this issue to achieve reliable and robust data output. Therefore, a long-term LOC-SERS measurement was carried over a time period of 40 000 s. For the measurement a 16 mm  25 mm all-glass microfluidic device equipped with six dosing nozzles for injecting the required reagents, a meandering mixing channel, and a long measuring channel loop to vary the aggregation time was applied. For the injection of required reagents a neMESYS high-precision syringe pump system (from Cetoni) was connected to the LOC device by 1/16 in. PEEK capillaries with an inner diameter of 0.25 mm for the analyte stream and 1/16 in. PTFE capillaries with an inner diameter of 0.5 mm for the other reagents. With the help of the syringe pump system, the injection of reagents can be set and controlled individually for each solution. The microfluidic LOC device was especially designed for SERS measurements and is described previously in more detail. 7 To provide the highest possible reproducibility with regard to the measurement principle, a segmented flow was created. In doing so, mineral oil was used as a separation medium and injected into the main microfluidic channel with a flow rate of 0.01 μL/s. The other reagents applied for the investigation reported here were water-soluble and formed droplets within the mineral oil as shown in Figure 1. Received: June 21, 2011 Accepted: September 14, 2011 ABSTRACT: Concerning the usability of lab-on-a-chip surface enhanced Raman spectroscopy (LOC-SERS) for analytical tasks applying chemometric data evalutation, a secure, repro- ducible, and stable data output independent of inconsistent ambient conditions has to be accomplished. In this contribu- tion, we present a new approach to achieve reliable and robust measurements based on segmented flow LOC-SERS via online- wavenumber calibration.