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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.
- 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.
- ItemProfiling of Saharan dust from the Caribbean to western Africa-Part 1: Layering structures and optical properties from shipborne polarization/Raman lidar observations(Katlenburg-Lindau : EGU, 2017) Rittmeister, Franziska; Ansmann, Albert; Engelmann, Ronny; Skupin, Annett; Baars, Holger; Kanitz, Thomas; Kinne, StefanWe present final and quality-assured results of multiwavelength polarization/Raman lidar observations of the Saharan air layer (SAL) over the tropical Atlantic. Observations were performed aboard the German research vessel R/V Meteor during the 1-month transatlantic cruise from Guadeloupe to Cabo Verde over 4500 km from 61.5 to 20 W at 14-15 N in April-May 2013. First results of the shipborne lidar measurements, conducted in the framework of SALTRACE (Saharan Aerosol Long-range Transport and Aerosol-Cloud Interaction Experiment), were reported by Kanitz et al. (2014). Here, we present four observational cases representing key stages of the SAL evolution between Africa and the Caribbean in detail in terms of layering structures and optical properties of the mixture of predominantly dust and aged smoke in the SAL. We discuss to what extent the lidar results confirm the validity of the SAL conceptual model which describes the dust long-range transport and removal processes over the tropical Atlantic. Our observations of a clean marine aerosol layer (MAL, layer from the surface to the SAL base) confirm the conceptual model and suggest that the removal of dust from the MAL, below the SAL, is very efficient. However, the removal of dust from the SAL assumed in the conceptual model to be caused by gravitational settling in combination with large-scale subsidence is weaker than expected. To explain the observed homogenous (heightindependent) dust optical properties from the SAL base to the SAL top, from the African coast to the Caribbean, we have to assume that the particle sedimentation strength is reduced and dust vertical mixing and upward transport mechanisms must be active in the SAL. Based on lidar observations on 20 nights at different longitudes in May 2013, we found, on average, MAL and SAL layer mean values (at 532 nm) of the extinction-to-backscatter ratio (lidar ratio) of 17-5 sr (MAL) and 43±8 sr (SAL), of the particle linear depolarization ratio of 0:025±0:015 (MAL) and 0:19±0:09 (SAL), and of the particle extinction coefficient of 67±45Mm..1 (MAL) and 68±37Mm..1 (SAL). The 532 nm optical depth of the lofted SAL was found to be, on average, 0:15±0:13 during the ship cruise. The comparably low values of the SAL mean lidar ratio and depolarization ratio (compared to typical pure dust values of 50-60 sr and 0.3, respectively) in combination with backward trajectories indicate a smoke contribution to light extinction of the order of 20% during May 2013, at the end of the burning season in central-western Africa. 1.
- 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.
- ItemQuantification of waves in lidar observations of noctilucent clouds at scales from seconds to minutes(Göttingen : Copernicus, 2013) Kaifler, N.; Baumgarten, G.; Fiedler, J.We present small-scale structures and waves observed in noctilucent clouds (NLC) by lidar at an unprecedented temporal resolution of 30 s or less. The measurements were taken with the Rayleigh/Mie/Raman lidar at the ALOMAR observatory in northern Norway (69 N) in the years 2008-2011. We find multiple layer NLC in 7.9% of the time for a brightness threshold of δ β 12 × 10-10 m-1 sr-1. In comparison to 10 min averaged data, the 30 s dataset shows considerably more structure. For limited periods, quasi-monochromatic waves in NLC altitude variations are common, in accord with ground-based NLC imagery. For the combined dataset, on the other hand, we do not find preferred periods but rather significant periods at all timescales observed (1 min to 1 h). Typical wave amplitudes in the layer vertical displacements are 0.2 km with maximum amplitudes up to 2.3 km. Average spectral slopes of temporal altitude and brightness variations are-2.01 ± 0.25 for centroid altitude,-1.41 ± 0.24 for peak brightness and-1.73 ± 0.25 for integrated brightness. Evaluating a new single-pulse detection system, we observe altitude variations of 70 s period and spectral slopes down to a scale of 10 s. We evaluate the suitability of NLC parameters as tracers for gravity waves.
- ItemDirect raman spectroscopic measurements of biological nitrogen fixation under natural conditions: An analytical approach for studying nitrogenase activity(Columbus, Ohio : American Chemical Society, 2016) Jochum, Tobias; Fastnacht, Agnes; Trumbore, Susan E.; Popp, Jürgen; Frosch, TorstenBiological N2 fixation is a major input of bioavailable nitrogen, which represents the most frequent factor limiting the agricultural production throughout the world. Especially, the symbiotic association between legumes and Rhizobium bacteria can provide substantial amounts of nitrogen (N) and reduce the need for industrial fertilizers. Despite its importance in the global N cycle, rates of biological nitrogen fixation have proven difficult to quantify. In this work, we propose and demonstrate a simple analytical approach to measure biological N2 fixation rates directly without a proxy or isotopic labeling. We determined a mean N2 fixation rate of 78 ± 5 μmol N2 (g dry weight nodule)-1 h-1 of a Medicago sativa-Rhizobium consortium by continuously analyzing the amount of atmospheric N2 in static environmental chambers with Raman gas spectroscopy. By simultaneously analyzing the CO2 uptake and photosynthetic plant activity, we think that a minimum CO2 mixing ratio might be needed for natural N2 fixation and only used the time interval above this minimum CO2 mixing ratio for N2 fixation rate calculations. The proposed approach relies only on noninvasive measurements of the gas phase and, given its simplicity, indicates the potential to estimate biological nitrogen fixation of legume symbioses not only in laboratory experiments. The same methods can presumably also be used to detect N2 fluxes by denitrification from ecosystems to the atmosphere. (Figure Presented).
- ItemA Machine Learning-Based Raman Spectroscopic Assay for the Identification of Burkholderia mallei and Related Species(Basel : MDPI, 2019) Silge, Anja; Moawad, Amira A.; Bocklitz, Thomas; Fischer, Katja; Rösch, Petra; Roesler, Uwe; Elschner, Mandy C.; Popp, Jürgen; Neubauer, HeinrichBurkholderia (B.) mallei, the causative agent of glanders, and B. pseudomallei, the causative agent of melioidosis in humans and animals, are genetically closely related. The high infectious potential of both organisms, their serological cross-reactivity, and similar clinical symptoms in human and animals make the differentiation from each other and other Burkholderia species challenging. The increased resistance against many antibiotics implies the need for fast and robust identification methods. The use of Raman microspectroscopy in microbial diagnostic has the potential for rapid and reliable identification. Single bacterial cells are directly probed and a broad range of phenotypic information is recorded, which is subsequently analyzed by machine learning methods. Burkholderia were handled under biosafety level 1 (BSL 1) conditions after heat inactivation. The clusters of the spectral phenotypes and the diagnostic relevance of the Burkholderia spp. were considered for an advanced hierarchical machine learning approach. The strain panel for training involved 12 B. mallei, 13 B. pseudomallei and 11 other Burkholderia spp. type strains. The combination of top- and sub-level classifier identified the mallei-complex with high sensitivities (>95%). The reliable identification of unknown B. mallei and B. pseudomallei strains highlighted the robustness of the machine learning-based Raman spectroscopic assay. © 2019 by the authors
- ItemCounterfeit and substandard test of the antimalarial tablet Riamet® by means of Raman hyperspectral multicomponent analysis(Basel : MDPI, 2019) Frosch, Timea; Wyrwich, Elisabeth; Yan, Di; Domes, Christian; Domes, Robert; Popp, Jürgen; Frosch, TorstenThe fight against counterfeit pharmaceuticals is a global issue of utmost importance, as failed medication results in millions of deaths every year. Particularly affected are antimalarial tablets. A very important issue is the identification of substandard tablets that do not contain the nominal amounts of the active pharmaceutical ingredient (API), and the differentiation between genuine products and products without any active ingredient or with a false active ingredient. This work presents a novel approach based on fiber-array based Raman hyperspectral imaging to qualify and quantify the antimalarial APIs lumefantrine and artemether directly and non-invasively in a tablet in a time-efficient way. The investigations were carried out with the antimalarial tablet Riamet® and self-made model tablets, which were used as examples of counterfeits and substandard. Partial least-squares regression modeling and density functional theory calculations were carried out for quantification of lumefantrine and artemether and for spectral band assignment. The most prominent differentiating vibrational signatures of the APIs were presented.