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Author: Krafft, Christoph × End date: 2019 × Start date: 2017 × Version: publishedVersion ×

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    Surface-enhanced Raman spectroscopy of cell lysates mixed with silver nanoparticles for tumor classification
    (Frankfurt, M. : Beilstein-Institut zur Förderung der Chemischen Wissenschaften, 2017) Hassoun, Mohamed; Schie, Iwan W.; Tolstik, Tatiana; Stanca, Sarmiza E.; Krafft, Christoph; Popp, Jürgen
    The throughput of spontaneous Raman spectroscopy for cell identification applications is limited to the range of one cell per second because of the relatively low sensitivity. Surface-enhanced Raman scattering (SERS) is a widespread way to amplify the intensity of Raman signals by several orders of magnitude and, consequently, to improve the sensitivity and throughput. SERS protocols using immuno-functionalized nanoparticles turned out to be challenging for cell identification because they require complex preparation procedures. Here, a new SERS strategy is presented for cell classification using non-functionalized silver nanoparticles and potassium chloride to induce aggregation. To demonstrate the principle, cell lysates were prepared by ultrasonication that disrupts the cell membrane and enables interaction of released cellular biomolecules to nanoparticles. This approach was applied to distinguish four cell lines – Capan-1, HepG2, Sk-Hep1 and MCF-7 – using SERS at 785 nm excitation. Six independent batches were prepared per cell line to check the reproducibility. Principal component analysis was applied for data reduction and assessment of spectral variations that were assigned to proteins, nucleotides and carbohydrates. Four principal components were selected as input for classification models based on support vector machines. Leave-three-batches-out cross validation recognized four cell lines with sensitivities, specificities and accuracies above 96%. We conclude that this reproducible and specific SERS approach offers prospects for cell identification using easily preparable silver nanoparticles.
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    The Staphylococcus aureus extracellular matrix protein (Emp) has a fibrous structure and binds to different extracellular matrices
    (Berlin : Nature Pulishing, 2017) Geraci, Jennifer; Neubauer, Svetlana; Pöllath, Christine; Hansen, Uwe; Rizzo, Fabio; Krafft, Christoph; Westermann, Martin; Hussain, Muzaffar; Peters, Georg; Pletz, Mathias W.; Löffler, Bettina; Makarewicz, Oliwia; Tuchscherr, Lorena
    The extracellular matrix protein Emp of Staphylococcus aureus is a secreted adhesin that mediates interactions between the bacterial surface and extracellular host structures. However, its structure and role in staphylococcal pathogenesis remain unknown. Using multidisciplinary approaches, including circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, transmission electron (TEM) and immunogold transmission electron microscopy, functional ELISA assays and in silico techniques, we characterized the Emp protein. We demonstrated that Emp and its truncated forms bind to suprastructures in human skin, cartilage or bone, among which binding activity seems to be higher for skin compounds. The binding domain is located in the C-terminal part of the protein. CD spectroscopy revealed high contents of β-sheets (39.58%) and natively disordered structures (41.2%), and TEM suggested a fibrous structure consisting of Emp polymers. The N-terminus seems to be essential for polymerization. Due to the uncommonly high histidine content, we suggest that Emp represents a novel type of histidine-rich protein sharing structural similarities to leucine-rich repeats proteins as predicted by the I-TASSER algorithm. These new findings suggest a role of Emp in infections of deeper tissue and open new possibilities for the development of novel therapeutic strategies.
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    Evaluation of shifted excitation Raman difference spectroscopy and comparison to computational background correction methods applied to biochemical Raman spectra
    (Basel : MDPI, 2017) Cordero, Eliana; Korinth, Florian; Stiebing, Clara; Krafft, Christoph; Schie, Iwan W.; Popp, Jürgen
    Raman spectroscopy provides label-free biochemical information from tissue samples without complicated sample preparation. The clinical capability of Raman spectroscopy has been demonstrated in a wide range of in vitro and in vivo applications. However, a challenge for in vivo applications is the simultaneous excitation of auto-fluorescence in the majority of tissues of interest, such as liver, bladder, brain, and others. Raman bands are then superimposed on a fluorescence background, which can be several orders of magnitude larger than the Raman signal. To eliminate the disturbing fluorescence background, several approaches are available. Among instrumentational methods shifted excitation Raman difference spectroscopy (SERDS) has been widely applied and studied. Similarly, computational techniques, for instance extended multiplicative scatter correction (EMSC), have also been employed to remove undesired background contributions. Here, we present a theoretical and experimental evaluation and comparison of fluorescence background removal approaches for Raman spectra based on SERDS and EMSC.