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- ItemSecondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies(Columbus, Ohio : American Chemical Society, 2016) Davies, Heather S.; Singh, Prabha; Deckert-Gaudig, Tanja; Deckert, Volker; Rousseau, Karine; Ridley, Caroline E.; Dowd, Sarah E.; Doig, Andrew J.; Pudney, Paul D. A.; Thornton, David J.; Blanch, Ewan W.The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUC5B. ROA indicated that the protein backbone of MUC5B is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUC5B O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUC5B (D1-D2-D′-D3 domains, NT5B), C-terminal region of MUC5B (D4-B-C-CK domains, CT5B) and the Cys-domain (within the central mucin domain of MUC5B) were found to be dominated by the β-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUC5B molecule. Interestingly, the molecule was found to contain a large amount of α-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin.
- ItemDisturbing-free determination of yeast concentration in DI water and in glucose using impedance biochips(Basel : MDPI AG, 2020) Kiani, M.; Du, N.; Vogel, M.; Raff, J.; Hübner, U.; Skorupa, I.; Bürger, D.; Schulz, S.E.; Schmidt, O.G.; Blaschke, D.; Schmidt, H.Deionized water and glucose without yeast and with yeast (Saccharomyces cerevisiae) of optical density OD600 that ranges from 4 to 16 has been put in the ring electrode region of six different types of impedance biochips and impedance has been measured in dependence on the added volume (20, 21, 22, 23, 24, 25 µL). The measured impedance of two out of the six types of biochips is strongly sensitive to the addition of both liquid without yeast and liquid with yeast and modelled impedance reveals a linear relationship between the impedance model parameters and yeast concentration. The presented biochips allow for continuous impedance measurements without interrupting the cultivation of the yeast. A multiparameter fit of the impedance model parameters allows for determining the concentration of yeast (cy) in the range from cy = 3.3 × 107 to cy = 17 × 107 cells/mL. This work shows that independent on the liquid, i.e., DI water or glucose, the impedance model parameters of the two most sensitive types of biochips with liquid without yeast and with liquid with yeast are clearly distinguishable for the two most sensitive types of biochips.
- ItemNon-instrumented DNA isolation, amplification and microarray-based hybridization for a rapid on-site detection of devastating Phytophthora kernoviae(Cambridge : Soc., 2015) Schwenkbier, Lydia; Pollok, Sibyll; Rudloff, Anne; Sailer, Sebastian; Cialla-May, Dana; Weber, Karina; Popp, JürgenA rapid and simple instrument-free detection system was developed for the identification of the plant pathogen Phytophthora kernoviae (P. kernoviae). The on-site operable analysis steps include magnetic particle based DNA isolation, helicase-dependent amplification (HDA) and chip-based DNA hybridization. The isothermal approach enabled the convenient amplification of the yeast GTP-binding protein (Ypt1) target gene in a miniaturized HDA-zeolite-heater (HZH) by an exothermic reaction. The amplicon detection on the chip was performed under room temperature conditions – either by successive hybridization and enzyme binding or by a combined step. A positive signal is displayed by enzymatically generated silver nanoparticle deposits, which serve as robust endpoint signals allowing an immediate visual readout. The hybridization assay enabled the reliable detection of 10 pg μL−1 target DNA. This is the first report of an entirely electricity-free, field applicable detection approach for devastating Phytophthora species, exemplarily shown for P. kernoviae.
- ItemLabel-free detection of Phytophthora ramorum using surface-enhanced Raman spectroscopy(Cambridge : Soc., 2015) Yüksel, Sezin; Schwenkbier, Lydia; Pollok, Sibyll; Weber, Karina; Cialla-May, Dana; Popp, JürgenIn this study, we report on a novel approach for the label-free and species-specific detection of the plant pathogen Phytophthora ramorum from real samples using surface enhanced Raman scattering (SERS). In this context, we consider the entire analysis chain including sample preparation, DNA isolation, amplification and hybridization on SERS substrate-immobilized adenine-free capture probes. Thus, the SERS-based detection of target DNA is verified by the strong spectral feature of adenine which indicates the presence of hybridized target DNA. This property was realized by replacing adenine moieties in the species-specific capture probes with 2-aminopurine. In the case of the matching capture and target sequence, the characteristic adenine peak serves as an indicator for specific DNA hybridization. Altogether, this is the first assay demonstrating the detection of a plant pathogen from an infected plant material by label-free SERS employing DNA hybridization on planar SERS substrates consisting of silver nanoparticles.