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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.
- ItemComparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study(Columbus, Ohio : American Chemical Society, 2020) Guo S.; Beleites C.; Neugebauer U.; Abalde-Cela S.; Afseth N.K.; Alsamad F.; Anand S.; Araujo-Andrade C.; Aškrabić S.; Avci E.; Baia M.; Baranska M.; Baria E.; Batista De Carvalho L.A.E.; De Bettignies P.; Bonifacio A.; Bonnier F.; Brauchle E.M.; Byrne H.J.; Chourpa I.; Cicchi R.; Cuisinier F.; Culha M.; Dahms M.; David C.; Duponchel L.; Duraipandian S.; El-Mashtoly S.F.; Ellis D.I.; Eppe G.; Falgayrac G.; Gamulin O.; Gardner B.; Gardner P.; Gerwert K.; Giamarellos-Bourboulis E.J.; Gizurarson S.; Gnyba M.; Goodacre R.; Grysan P.; Guntinas-Lichius O.; Helgadottir H.; Grošev V.M.; Kendall C.; Kiselev R.; Kölbach M.; Krafft C.; Krishnamoorthy S.; Kubryck P.; Lendl B.; Loza-Alvarez P.; Lyng F.M.; Machill S.; Malherbe C.; Marro M.; Marques M.P.M.; Matuszyk E.; Morasso C.F.; Moreau M.; Muhamadali H.; Mussi V.; Notingher I.; Pacia M.Z.; Pavone F.S.; Penel G.; Petersen D.; Piot O.; Rau J.V.; Richter M.; Rybarczyk M.K.; Salehi H.; Schenke-Layland K.; Schlücker S.; Schosserer M.; Schütze K.; Sergo V.; Sinjab F.; Smulko J.; Sockalingum G.D.; Stiebing C.; Stone N.; Untereiner V.; Vanna R.; Wieland K.; Popp J.; Bocklitz T.The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies. © 2020 American Chemical Society.
- ItemDistinction of nucleobases - A tip-enhanced Raman approach(Frankfurt, M. : Beilstein-Institut zur Förderung der Chemischen Wissenschaften, 2011) Treffer, R.; Lin, X.; Bailo, E.; Deckert-Gaudig, T.; Deckert, V.The development of novel DNA sequencing methods is one of the ongoing challenges in various fields of research seeking to address the demand for sequence information. However, many of these techniques rely on some kind of labeling or amplification steps. Here we investigate the intrinsic properties of tip-enhanced Raman scattering (TERS) towards the development of a novel, label-free, direct sequencing method. It is known that TERS allows the acquisition of spectral information with high lateral resolution and single-molecule sensitivity. In the presented experiments, single stranded adenine and uracil homopolymers were immobilized on different kinds of substrates (mica and gold nanoplates) and TERS experiments were conducted, which demonstrated the reproducibility of the technique. To elucidate the signal contributions from the specific nucleobases, TERS spectra were collected on single stranded calf thymus DNA with arbitrary sequence. The results show that, while the Raman signals with respect to the four nucleobases differ remarkably, specific markers can be determined for each respective base. The combination of sensitivity and reproducibility shows that the crucial demands for a sequencing procedure are met.
- ItemTowards multiple readout application of plasmonic arrays(Frankfurt, M. : Beilstein-Institut zur Förderung der Chemischen Wissenschaften, 2011) Cialla, D.; Weber, K.; Böhme, R.; Hübner, U.; Schneidewind, H.; Zeisberger, M.; Mattheis, R.; Möller, R.; Popp, J.In order to combine the advantages of fluorescence and surface-enhanced Raman spectroscopy (SERS) on the same chip platform, a nanostructured gold surface with a unique design, allowing both the sensitive detection of fluorescence light together with the specific Raman fingerprint of the fluorescent molecules, was established. This task requires the fabrication of plasmonic arrays that permit the binding of molecules of interest at different distances from the metallic surface. The most efficient SERS enhancement is achieved for molecules directly adsorbed on the metallic surface due to the strong field enhancement, but where, however, the fluorescence is quenched most efficiently. Furthermore, the fluorescence can be enhanced efficiently by careful adjustment of the optical behavior of the plasmonic arrays. In this article, the simultaneous application of SERS and fluorescence, through the use of various gold nanostructured arrays, is demonstrated by the realization of a DNA detection scheme. The results shown open the way to more flexible use of plasmonic arrays in bioanalytics.
- ItemOne year of Raman lidar observations of free-tropospheric aerosol layers over South Africa(München : European Geopyhsical Union, 2015) Giannakaki, E.; Pfüller, A.; Korhonen, K.; Mielonen, T.; Laakso, L.; Vakkari, V.; Baars, H.; Engelmann, R.; Beukes, J.P.; Van Zyl, P.G.; Josipovic, M.; Tiitta, P.; Chiloane, K.; Piketh, S.; Lihavainen, H.; Lehtinen, K.E.J.; Komppula, M.Raman lidar data obtained over a 1 year period has been analysed in relation to aerosol layers in the free troposphere over the Highveld in South Africa. In total, 375 layers were observed above the boundary layer during the period 30 January 2010 to 31 January 2011. The seasonal behaviour of aerosol layer geometrical characteristics, as well as intensive and extensive optical properties were studied. The highest centre heights of free-tropospheric layers were observed during the South African spring (2520 ± 970 m a.g.l., also elsewhere). The geometrical layer depth was found to be maximum during spring, while it did not show any significant difference for the rest of the seasons. The variability of the analysed intensive and extensive optical properties was high during all seasons. Layers were observed at a mean centre height of 2100 ± 1000 m with an average lidar ratio of 67 ± 25 sr (mean value with 1 standard deviation) at 355 nm and a mean extinction-related Ångström exponent of 1.9 ± 0.8 between 355 and 532 nm during the period under study. Except for the intensive biomass burning period from August to October, the lidar ratios and Ångström exponents are within the range of previous observations for urban/industrial aerosols. During Southern Hemispheric spring, the biomass burning activity is clearly reflected in the optical properties of the observed free-tropospheric layers. Specifically, lidar ratios at 355 nm were 89 ± 21, 57 ± 20, 59 ± 22 and 65 ± 23 sr during spring (September–November), summer (December–February), autumn (March–May) and winter (June–August), respectively. The extinction-related Ångström exponents between 355 and 532 nm measured during spring, summer, autumn and winter were 1.8 ± 0.6, 2.4 ± 0.9, 1.8 ± 0.9 and 1.8 ± 0.6, respectively. The mean columnar aerosol optical depth (AOD) obtained from lidar measurements was found to be 0.46 ± 0.35 at 355 nm and 0.25 ± 0.2 at 532 nm. The contribution of free-tropospheric aerosols on the AOD had a wide range of values with a mean contribution of 46%.
- ItemTechnical Note: One year of Raman-lidar measurements in Gual Pahari EUCAARI site close to New Delhi in India – Seasonal characteristics of the aerosol vertical structure(München : European Geopyhsical Union, 2012) Komppula, M.; Mielonen, T.; Arola, A.; Korhonen, K.; Lihavainen, H.; Hyvärinen, A.-P.; Baars, H.; Engelmann, R.; Althausen, D.; Ansmann, A.; Müller, D.; Panwar, T.S.; Hooda, R.K.; Sharma, V.P.; Kerminen, V.-M.; Lehtinen, K.E.J.; Viisanen, Y.One year of multi-wavelength (3 backscatter + 2 extinction + 1 depolarization) Raman lidar measurements at Gual Pahari, close to New Delhi, were analysed. The data was split into four seasons: spring (March–May), summer (June–August), autumn (September–November) and winter (December–February). The vertical profiles of backscatter, extinction, and lidar ratio and their variability during each season are presented. The measurements revealed that, on average, the aerosol layer was at its highest in spring (5.5 km). In summer, the vertically averaged (between 1–3 km) backscatter and extinction coefficients had the highest averages (3.3 Mm−1 sr−1 and 142 Mm−1 at 532 nm, respectively). Aerosol concentrations were slightly higher in summer compared to other seasons, and particles were larger in size. The autumn showed the highest lidar ratio and high extinction-related Ångström exponents (AEext), indicating the presence of smaller probably absorbing particles. The winter had the lowest backscatter and extinction coefficients, but AEext was the highest, suggesting still a large amount of small particles.
- ItemSurface-Enhanced Raman Spectroscopy to Characterize Different Fractions of Extracellular Vesicles from Control and Prostate Cancer Patients(Basel : MDPI, 2021) Osei, Eric Boateng; Paniushkina, Liliia; Wilhelm, Konrad; Popp, Jürgen; Nazarenko, Irina; Krafft, ChristophExtracellular vesicles (EVs) are membrane-enclosed structures ranging in size from about 60 to 800 nm that are released by the cells into the extracellular space; they have attracted interest as easily available biomarkers for cancer diagnostics. In this study, EVs from plasma of control and prostate cancer patients were fractionated by differential centrifugation at 5000× g, 12,000× g and 120,000× g. The remaining supernatants were purified by ultrafiltration to produce EV-depleted free-circulating (fc) fractions. Spontaneous Raman and surface-enhanced Raman spectroscopy (SERS) at 785 nm excitation using silver nanoparticles (AgNPs) were employed as label-free techniques to collect fingerprint spectra and identify the fractions that best discriminate between control and cancer patients. SERS spectra from 10 µL droplets showed an enhanced Raman signature of EV-enriched fractions that were much more intense for cancer patients than controls. The Raman spectra of dehydrated pellets of EV-enriched fractions without AgNPs were dominated by spectral contributions of proteins and showed variations in S-S stretch, tryptophan and protein secondary structure bands between control and cancer fractions. We conclude that the AgNPs-mediated SERS effect strongly enhances Raman bands in EV-enriched fractions, and the fractions, EV12 and EV120 provide the best separation of cancer and control patients by Raman and SERS spectra.
- ItemCeilometer lidar comparison: Backscatter coefficient retrieval and signal-to-noise ratio determination(München : European Geopyhsical Union, 2010) Heese, B.; Flentje, H.; Althausen, D.; Ansmann, A.; Frey, S.The potential of a new generation of ceilometer instruments for aerosol monitoring has been studied in the Ceilometer Lidar Comparison (CLIC) study. The used ceilometer was developed by Jenoptik, Germany, and is designed to find both thin cirrus clouds at tropopause level and aerosol layers at close ranges during day and night-time. The comparison study was performed to determine up to which altitude the ceilometers are capable to deliver particle backscatter coefficient profiles. For this, the derived ceilometer profiles are compared to simultaneously measured lidar profiles at the same wavelength. The lidar used for the comparison was the multi-wavelengths Raman lidar PollyXT. To demonstrate the capabilities and limits of ceilometers for the derivation of particle backscatter coefficient profiles from their measurements two examples of the comparison results are shown. Two cases, a daytime case with high background noise and a less noisy night-time case, are chosen. In both cases the ceilometer profiles compare well with the lidar profiles in atmospheric structures like aerosol layers or the boundary layer top height. However, the determination of the correct magnitude of the particle backscatter coefficient needs a calibration of the ceilometer data with an independent measurement of the aerosol optical depth by a sun photometer. To characterizes the ceilometers signal performance with increasing altitude a comprehensive signal-to-noise ratio study was performed. During daytime the signal-to-noise ratio is higher than 1 up to 4–5 km depending on the aerosol content. In our night-time case the SNR is higher than 1 even up to 8.5 km, so that also aerosol layers in the upper troposphere had been detected by the ceilometer.
- ItemCoincident measurements of PMSE and NLC above ALOMAR (69° N, 16° E) by radar and lidar from 1999-2008(Göttingen : Copernicus, 2011) Kaifler, N.; Baumgarten, G.; Fiedler, J.; Latteck, R.; Lübken, F.-J.; Rapp, M.Polar Mesosphere Summer Echoes (PMSE) and Noctilucent Clouds (NLC) have been routinely measured at the ALOMAR research facility in Northern Norway (69° N, 16° E) by lidar and radar, respectively. 2900 h of lidar measurements by the ALOMAR Rayleigh/Mie/Raman lidar were combined with almost 18 000 h of radar measurements by the ALWIN VHF radar, all taken during the years 1999 to 2008, to study simultaneous and common-volume observations of both phenomena. PMSE and NLC are known from both theory and observations to be positively linked. We quantify the occurrences of PMSE and/or NLC and relations in altitude, especially with respect to the lower layer boundaries. The PMSE occurrence rate is with 75.3% considerably higher than the NLC occurrence rate of 19.5%. For overlapping PMSE and NLC observations, we confirm the coincidence of the lower boundaries and find a standard deviation of 1.26 km, hinting at very fast sublimation rates. However, 10.1% of all NLC measurements occur without accompanying PMSE. Comparison of occurrence rates with solar zenith angle reveals that NLC without PMSE mostly occur around midnight indicating that the ice particles were not detected by the radar due to the reduced electron density.