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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.
- ItemDetection of Protein Glycosylation Using Tip-Enhanced Raman Scattering(Columbus, Ohio : American Chemical Society, 2016) Cowcher, David P.; Deckert-Gaudig, Tanja; Brewster, Victoria L.; Ashton, Lorna; Deckert, Volker; Goodacre, RoystonThe correct glycosylation of biopharmaceutical glycoproteins and their formulations is essential for them to have the desired therapeutic effect on the patient. It has recently been shown that Raman spectroscopy can be used to quantify the proportion of glycosylated protein from mixtures of native and glycosylated forms of bovine pancreatic ribonuclease (RNase). Here we show the first steps toward not only the detection of glycosylation status but the characterization of glycans themselves from just a few protein molecules at a time using tip-enhanced Raman scattering (TERS). While this technique generates complex data that are very dependent on the protein orientation, with the careful development of combined data preprocessing, univariate and multivariate analysis techniques, we have shown that we can distinguish between the native and glycosylated forms of RNase. Many glycoproteins contain populations of subtly different glycoforms; therefore, with stricter orientation control, we believe this has the potential to lead to further glycan characterization using TERS, which would have use in biopharmaceutical synthesis and formulation research.
- ItemModified bibenzimidazole ligands as spectator ligands in photoactive molecular functional Ru-polypyridine units? Implications from spectroscopy(Cambridge : RSC, 2014) Meyer-Ilse, J.; Bauroth, S.; Bräutigam, M.; Schmitt, M.; Popp, J.; Beckert, R.; Rockstroh, N.; Pilz, T.D.; Monczak, K.; Heinemann, F.W.; Rau, S.; Dietzek, B.The photophysical properties of Ruthenium-bipyridine complexes bearing a bibenzimidazole ligand were investigated. The nitrogens on the bibenzimidazole-ligand were protected, by adding either a phenylene group or a 1,2-ethandiyl group, to remove the photophysical dependence of the complex on the protonation state of the bibenzimidazole ligand. This protection results in the bibenzimidazole ligand contributing to the MLCT transition, which is experimentally evidenced by (resonance) Raman scattering in concert with DFT calculations for a detailed mode assignment in the (resonance) Raman spectra.
- ItemFemtosecond stimulated Raman spectroscopy of the cyclobutane thymine dimer repair mechanism: A computational study(Washington, DC : American Chemical Society, 2014) Ando, H.; Fingerhut, B.P.; Dorfman, K.E.; Biggs, J.D.; Mukamel, S.Cyclobutane thymine dimer, one of the major lesions in DNA formed by exposure to UV sunlight, is repaired in a photoreactivation process, which is essential to maintain life. The molecular mechanism of the central step, i.e., intradimer C-C bond splitting, still remains an open question. In a simulation study, we demonstrate how the time evolution of characteristic marker bands (C=O and C=C/C-C stretch vibrations) of cyclobutane thymine dimer and thymine dinucleotide radical anion, thymidylyl(3′→5′)-thymidine, can be directly probed with femtosecond stimulated Raman spectroscopy (FSRS). We construct a DFT(M05-2X) potential energy surface with two minor barriers for the intradimer C5-C′5 splitting and a main barrier for the C6-C′6 splitting, and identify the appearance of two C5=C6 stretch vibrations due to the C6-C′6 splitting as a spectroscopic signature of the underlying bond splitting mechanism. The sequential mechanism shows only absorptive features in the simulated FSRS signals, whereas the fast concerted mechanism shows characteristic dispersive line shapes. (Figure Presented).
- ItemLocal protonation control using plasmonic activation(Cambridge : RSC, 2001) Singh, P.; Deckert, V.Localized protonation of 4-mercaptopyridine (4-MPY), activated by light in the presence of silver nanoparticles is monitored under ambient conditions using surface-enhanced Raman scattering (SERS) and tip-enhanced Raman scattering (TERS). The reaction can be controlled by the excitation wavelength and the atmospheric conditions, thus, providing a tool for site-specific control of protonation.
- ItemFiber enhanced Raman spectroscopic analysis as a novel method for diagnosis and monitoring of diseases related to hyperbilirubinemia and hyperbiliverdinemia(Cambridge : Soc., 2016) Yan, Di; Domes, Christian; Domes, Robert; Frosch, Timea; Popp, Jürgen; Pletz, Mathias W.; Frosch, TorstenFiber enhanced resonance Raman spectroscopy (FERS) is introduced for chemically selective and ultrasensitive analysis of the biomolecules hematin, hemoglobin, biliverdin, and bilirubin. The abilities for analyzing whole intact, oxygenated erythrocytes are proven, demonstrating the potential for the diagnosis of red blood cell related diseases, such as different types of anemia and hemolytic disorders. The optical fiber enables an efficient light-guiding within a miniaturized sample volume of only a few micro-liters and provides a tremendously improved analytical sensitivity (LODs of 0.5 μM for bilirubin and 0.13 μM for biliverdin with proposed improvements down to the pico-molar range). FERS is a less invasive method than the standard ones and could be a new analytical method for monitoring neonatal jaundice, allowing a precise control of the unconjugated serum bilirubin levels, and therefore, providing a better prognosis for newborns. The potential for sensing very low concentrations of the bile pigments may also open up new opportunities for cancer research. The abilities of FERS as a diagnostic tool are explored for the elucidation of jaundice with different etiologies including the rare, not yet well understood diseases manifested in green jaundice. This is demonstrated by quantifying clinically relevant concentrations of bilirubin and biliverdin simultaneously in the micro-molar range: for the case of hyperbilirubinemia due to malignancy, infectious hepatitis, cirrhosis or stenosis of the common bile duct (1 μM biliverdin together with 50 μM bilirubin) and for hyperbiliverdinemia (25 μM biliverdin and 75 μM bilirubin). FERS has high potential as an ultrasensitive analytical technique for a wide range of biomolecules and in various life-science applications.
- ItemToward food analytics: fast estimation of lycopene and β-carotene content in tomatoes based on surface enhanced Raman spectroscopy (SERS)(Cambridge : Soc., 2016) Radu, Andreea Ioana; Ryabchykov, Oleg; Bocklitz, Thomas Wilhelm; Huebner, Uwe; Weber, Karina; Cialla-May, Dana; Popp, JürgenCarotenoids are molecules that play important roles in both plant development and in the well-being of mammalian organisms. Therefore, various studies have been performed to characterize carotenoids’ properties, distribution in nature and their health benefits upon ingestion. Nevertheless, there is a gap regarding a fast detection of them at the plant phase. Within this contribution we report the results obtained regarding the application of surface enhanced Raman spectroscopy (SERS) toward the differentiation of two carotenoid molecules (namely, lycopene and β-carotene) in tomato samples. To this end, an e-beam lithography (EBL) SERS-active substrate and a 488 nm excitation source were employed, and a relevant simulated matrix was prepared (by mixing the two carotenoids in defined percentages) and measured. Next, carotenoids were extracted from tomato plants and measured as well. Finally, a combination of principal component analysis and partial least squares regression (PCA-PLSR) was applied to process the data, and the obtained results were compared with HPLC measurements of the same extracts. A good agreement was obtained between the HPLC and the SERS results for most of the tomato samples.
- ItemHighly sensitive and specific detection of E. coli by a SERS nanobiosensor chip utilizing metallic nanosculptured thin films(Cambridge : Soc., 2015) Srivastava, Sachin K.; Hamo, Hilla Ben; Kushmaro, Ariel; Marks, Robert S.; Grüner, Christoph; Rauschenbach, Bernd; Abdulhalim, IbrahimA nanobiosensor chip, utilizing surface enhanced Raman spectroscopy (SERS) on nanosculptured thin films (nSTFs) of silver, was shown to detect Escherichia coli (E. coli) bacteria down to the concentration level of a single bacterium. The sensor utilizes highly enhanced plasmonic nSTFs of silver on a silicon platform for the enhancement of Raman bands as checked with adsorbed 4-aminothiophenol molecules. T-4 bacteriophages were immobilized on the aforementioned surface of the chip for the specific capture of target E. coli bacteria. To demonstrate that no significant non-specific immobilization of other bacteria occurs, three different, additional bacterial strains, Chromobacterium violaceum, Paracoccus denitrificans and Pseudomonas aeruginosa were used. Furthermore, experiments performed on an additional strain of E. coli to address the specificity and reusability of the sensor showed that the sensor operates for different strains of E. coli and is reusable. Time resolved phase contrast microscopy of the E. coli-T4 bacteriophage chip was performed to study its interaction with bacteria over time. Results showed that the present sensor performs a fast, accurate and stable detection of E. coli with ultra-small concentrations of bacteria down to the level of a single bacterium in 10 μl volume of the sample.
- ItemMicrobial respiration and natural attenuation of benzene contaminated soils investigated by cavity enhanced Raman multi-gas spectroscopy(Cambridge : Soc., 2015) Jochum, Tobias; Michalzik, Beate; Bachmann, Anne; Popp, Jürgen; Frosch, TorstenSoil and groundwater contamination with benzene can cause serious environmental damage. However, many soil microorganisms are capable to adapt and are known to strongly control the fate of organic contamination. Innovative cavity enhanced Raman multi-gas spectroscopy (CERS) was applied to investigate the short-term response of the soil micro-flora to sudden surface contamination with benzene regarding the temporal variations of gas products and their exchange rates with the adjacent atmosphere. 13C-labeled benzene was spiked on a silty-loamy soil column in order to track and separate the changes in heterotrophic soil respiration – involving 12CO2 and O2 – from the natural attenuation process of benzene degradation to ultimately form 13CO2. The respiratory quotient (RQ) decreased from a value 0.98 to 0.46 directly after the spiking and increased again within 33 hours to a value of 0.72. This coincided with the maximum 13CO2 concentration rate (0.63 μmol m−2 s−1), indicating the highest benzene degradation at 33 hours after the spiking event. The diffusion of benzene in the headspace and the biodegradation into 13CO2 were simultaneously monitored and 12 days after the benzene spiking no measurable degradation was detected anymore. The RQ finally returned to a value of 0.96 demonstrating the reestablished aerobic respiration.
- ItemOnline investigation of respiratory quotients in Pinus sylvestris and Picea abies during drought and shading by means of cavity-enhanced Raman multi-gas spectrometry(Cambridge : Soc., 2015) Hanf, Stefan; Fischer, Sarah; Hartmann, Henrik; Keiner, Robert; Trumbore, Susan; Popp, Jürgen; Frosch, TorstenPhotosynthesis and respiration are major components of the plant carbon balance. During stress, like drought, carbohydrate supply from photosynthesis is reduced and the Krebs cycle respiration must be fueled with other stored carbon compounds. However, the dynamics of storage use are still unknown. The respiratory quotient (RQ, CO2 released per O2 consumed during respiration) is an excellent indicator of the nature of the respiration substrate. In plant science, however, online RQ measurements have been challenging or even impossible so far due to very small gas exchange fluxes during respiration. Here we apply cavity-enhanced multi-gas Raman spectrometry (CERS) for online in situ RQ measurements in drought-tolerant pine (Pinus sylvestris [L.]) and drought-intolerant spruce (Picea abies [L. H. Karst]). Two different treatments, drought and shading, were applied to reduce photosynthesis and force dependency on stored substrates. Changes in respiration rates and RQ values were continuously monitored over periods of several days with low levels of variance. The results show that both species switched from COH-dominated respiration (RQ = 1.0) to a mixture of substrates during shading (RQ = 0.77–0.81), while during drought only pine did so (RQ = 0.75). The gas phase measurements were complemented by concentration measurements of non-structural carbohydrates and lipids. These first results suggest a physiological explanation for greater drought tolerance in pine. CERS was proven as powerful technique for non-consumptive and precise real-time monitoring of respiration rates and respirational quotients for the investigation of plant metabolism under drought stress conditions that are predicted to increase with future climate change.