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    Rapid isolation and identification of pneumonia associated pathogens from sputum samples combining an innovative sample preparation strategy and array-based detection
    (Washington : American Chemical Society, 2019) Pahlow, Susanne; Lehniger, Lydia; Hentschel, Stefanie; Seise, Barbara; Braun, Sascha D.; Ehricht, Ralf; Berg, Albrecht; Popp, Jürgen; Weber, Karina
    With this study, an innovative and convenient enrichment and detection strategy for eight clinically relevant pneumonia pathogens, namely, Acinetobacter baumannii, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae is introduced. Bacteria were isolated from sputum samples with amine-modified particles exploiting pH-dependent electrostatic interactions between bacteria and the functionalized particle surface. Following this, an asymmetric polymerase chain reaction as well as subsequent stringent array-based hybridization with specific complementary capture probes were performed. Finally, results were visualized by an enzyme-induced silver nanoparticle deposition, providing stable endpoint signals and consequently an easy detection possibility. The assay was optimized using spiked samples of artificial sputum with different strains of the abovementioned bacterial species. Furthermore, actual patient sputum samples with S. pneumoniae were successfully analyzed. The presented approach offers great potential for the urgent need of a fast, specific, and reliable isolation and identification platform for important pneumonia pathogens, covering the complete process chain from sample preparation up to array-based detection within only 4 h.With this study, an innovative and convenient enrichment and detection strategy for eight clinically relevant pneumonia pathogens, namely, Acinetobacter baumannii, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae is introduced. Bacteria were isolated from sputum samples with amine-modified particles exploiting pH-dependent electrostatic interactions between bacteria and the functionalized particle surface. Following this, an asymmetric polymerase chain reaction as well as subsequent stringent array-based hybridization with specific complementary capture probes were performed. Finally, results were visualized by an enzyme-induced silver nanoparticle deposition, providing stable endpoint signals and consequently an easy detection possibility. The assay was optimized using spiked samples of artificial sputum with different strains of the abovementioned bacterial species. Furthermore, actual patient sputum samples with S. pneumoniae were successfully analyzed. The presented approach offers great potential for the urgent need of a fast, specific, and reliable isolation and identification platform for important pneumonia pathogens, covering the complete process chain from sample preparation up to array-based detection within only 4 h.
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    UV absorption spectroscopy in water-filled antiresonant hollow core fibers for pharmaceutical detection
    (Basel : MDPI, 2018) Nissen, Mona; Doherty, Brenda; Hamperl, J.; Kobelke, Jens; Weber, Karina; Henkel, Thomas; Schmidt, Markus A.
    Due to a worldwide increased use of pharmaceuticals and, in particular, antibiotics, a growing number of these substance residues now contaminate natural water resources and drinking supplies. This triggers a considerable demand for low-cost, high-sensitivity methods for monitoring water quality. Since many biological substances exhibit strong and characteristic absorption features at wavelengths shorter than 300 nm, UV spectroscopy presents a suitable approach for the quantitative identification of such water-contaminating species. However, current UV spectroscopic devices often show limited light-matter interaction lengths, demand sophisticated and bulky experimental infrastructure which is not compatible with microfluidics, and leave large fractions of the sample analyte unused. Here, we introduce the concept of UV spectroscopy in liquid-filled anti-resonant hollow core fibers, with large core diameters and lengths of approximately 1 m, as a means to overcome such limitations. This extended light-matter interaction length principally improves the concentration detection limit by two orders of magnitude while using almost the entire sample volume—that is three orders of magnitude smaller compared to cuvette based approaches. By integrating the fibers into an optofluidic chip environment and operating within the lowest experimentally feasible transmission band, concentrations of the application-relevant pharmaceutical substances, sulfamethoxazole (SMX) and sodium salicylate (SS), were detectable down to 0.1 µM (26 ppb) and 0.4 µM (64 ppb), respectively, with the potential to reach significantly lower detection limits for further device integration.
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    TopUp SERS substrates with integrated internal standard
    (Basel : MDPI, 2018) Patze, Sophie; Hübner, Uwe; Weber, Karina; Cialla-May, Dana; Popp, Jürgen
    Surface-enhanced Raman spectroscopy (SERS) is known as a molecular-specific and highly sensitive method. In order to enable the routine application of SERS, powerful SERS substrates are of great importance. Within this manuscript, a TopUp SERS substrate is introduced which is fabricated by a top-down process based on microstructuring as well as a bottom-up generation of silver nanostructures. The Raman signal of the support material acts as an internal standard in order to improve the quantification capabilities. The analyte molecule coverage of sulfamethoxazole on the surface of the nanostructures is characterized by the SERS signal evolution fitted by a Langmuir–Freundlich isotherm.
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    Molecular Specific and Sensitive Detection of Pyrazinamide and Its Metabolite Pyrazinoic Acid by Means of Surface Enhanced Raman Spectroscopy Employing In Situ Prepared Colloids
    (Basel : MDPI, 2019) Mühlig, Anna; Jahn, Izabella-Jolan; Heidler, Jan; Weber, Karina; Jahn, Martin; Sheen, Patricia; Zimic, Mirko; Cialla-May, Dana; Popp, Jürgen
    The prodrug pyrazinamide (PZA) is metabolized by the mycobacteria to pyrazinoic acid (POA), which is expelled into the extracellular environment. PZA resistance is highly associated to a lack of POA efflux. Thus, by detecting a reduction of the concentration of POA in the extracellular environment, by means of lab-on-a-chip (LoC)-SERS (surface-enhanced Raman spectroscopy), an alternative approach for the discrimination of PZA resistant mycobacteria is introduced. A droplet-based microfluidic SERS device has been employed to illustrate the potential of the LoC-SERS method for the discrimination of PZA resistant mycobacteria. The two analytes were detected discretely in aqueous solution with a limit of detection of 27 µm for PZA and 21 µm for POA. The simultaneous detection of PZA and POA in aqueous mixtures could be realized within a concentration range from 20 μm to 50 μm for PZA and from 50 μm to 80 μm for POA.