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Microfluidic paper-based analytical devices (μPADs) in combination with surface enhanced Raman scattering (SERS) provide a way for analyses of complex mixtures. The μPADs can be used for the chromatographic separation of different compounds of mixtures in combination with the separate detection of the analytes in different zones on the paper by SERS. SERS allows to observe analytes directly without labelling in low concentrations in aqueous solutions and to identify them by their spectral fingerprint.
SERS substrates on the μPADs were created by drying standard silver nanoparticle (AgNP) solution on the paper. The microfluidic structure of the μPADs was prepared by wax printing. As a model system, an aqueous solution of the non-fluorescent analyte adenine and two fluorescent dyes tris(2,2’-bipyridyl)dichlororuthenium(II) and sulforhodamine B was tested. The dependency of the SERS signal intensity on the analyte concentration can be fitted using a Langmuir isotherm curve progression. With this approach, a semi-quantitative analysis of the components is possible. The reproducibility and stability of the measurement procedure was tested with several measurements over time, different NP batches, and with different analytes in different concentrations and resulted in an average relative standard deviation of 16 %.
The SERS spectra of the mixture of the model system are dominated by one compound depending on the concentration ratio. For the detection and identification of all components of the mixture, the compounds were therefore separated on the μPADs and measured at different positions. The position of adenine on the μPADs is dependent on the AgNP coverage of the paper. Due to this effect, it possible to detect adenine on a defined point on the μPADs and to get an information on the concentration in a mixture of three components.
The growing need to implement sensors such as NIR or Raman spectroscopy for the in-situ monitoring of bioprocesses which follows the standards of Quality by Design is either restricted by the impact of the huge water signal or by a disturbing fluorescence background originating from compounds in the culture media. Furthermore, the characterization of the bioprocess samples is challenging due to changing conditions in course of cultivation.
Here we evaluate two different process-suitable Raman spectroscopic approaches, namely time-gated Raman which bears the potential to extract the Raman signal from the fluorescence background, and cw- Raman with NIR excitation in combination with Surface Enhanced Raman Spectroscopy- (SERS) to investigate cell-free supernatants of Escherichia coli sampled over the course of a cultivation. A confocal Raman microscope was used as a reference for the process devices. The concentration of the analytes, glucose, acetate as well as metabolites such as cAMP, AMP and amino-acids were determined by offline by High-Performance Liquid Chromatography (HPLC) to serve as reference for the calibration of the Raman and SERS spectral data.
Multivariate evaluation of the Raman and SERS spectra by Partial Least Squares Regression (PLSR) yielded for most of the analytes robust correlations at each sampling point. Repeated investigation of the off-line samples over a larger experimental period suggested not only a high reliability of the Raman data in general but also a high repeatability of the SERS experiments. Similar spectral features in different quality and signal/noise ratios were measured with all three set-ups. Major results of the comparison of the different Raman spectroscopic approaches and their combination with SERS are summarized and conclusions are drawn on which approach provides the most accurate concentration data among the target analytes.
Acknowledgement
The authors kindly thank Mario Birkholz (IHP, Frankfurt (Oder), Germany) for the opportunity to use a confocal Raman microscope, Alex Bunker and Tapani Viitala (Division of Pharmaceutical Biosciences, Centre for Drug Research, University of Helsinki, Finland).