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- ItemRapid Colorimetric Detection of Pseudomonas aeruginosa in Clinical Isolates Using a Magnetic Nanoparticle Biosensor(Washington, DC : ACS Publications, 2019) Alhogail, Sahar; Suaifan, Ghadeer A.R.Y; Bikker, Floris J.; Kaman, Wendy E.; Weber, Karina; Cialla-May, Dana; Popp, Jürgen; Zourob, Mohammed M.A rapid, sensitive, and specific colorimetric biosensor based on the use of magnetic nanoparticles (MNPs) was designed for the detection of Pseudomonas aeruginosa in clinical samples. The biosensing platform was based on the measurement of P. aeruginosa proteolytic activity using a specific protease substrate. At the N-terminus, this substrate was covalently bound to MNPs and was linked to a gold sensor surface via cystine at the C-terminus of the substrates. The golden sensor appears black to naked eyes because of the coverage of the MNPs. However, upon proteolysis, the cleaved peptide–MNP moieties will be attracted by an external magnet, revealing the golden color of the sensor surface, which can be observed by the naked eye. In vitro, the biosensor was able to detect specifically and quantitatively the presence of P. aeruginosa with a detection limit of 102 cfu/mL in less than 1 min. The colorimetric biosensor was used to test its ability to detect in situ P. aeruginosa in clinical isolates from patients. This biochip is anticipated to be useful as a rapid point-of-care device for the diagnosis of P. aeruginosa-related infections.
- ItemTowards on-site testing of Phytophthora species(Cambridge : RSC Publ., 2014) Schwenkbier, Lydia; Pollok, Sibyll; König, Stephan; Urban, Matthias; Werres, Sabine; Cialla-May, Dana; Weber, Karina; Popp, JürgenRapid detection and accurate identification of plant pathogens in the field is an ongoing challenge. In this study, we report for the first time on the development of a helicase-dependent isothermal amplification (HDA) in combination with on-chip hybridization for the detection of selected Phytophthora species. The HDA approach allows efficient amplification of the yeast GTP-binding protein (Ypt1) target gene region at one constant temperature in a miniaturized heating device. The assay's specificity was determined by on-chip DNA hybridization and subsequent silver nanoparticle deposition. The silver deposits serve as stable endpoint signals that enable the visual as well as the electrical readout. Our promising results point to the direction of a near future on-site application of the combined techniques for a reliable detection of Phytophthora species.
- 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.
- ItemFiber-based SORS-SERDS system and chemometrics for the diagnostics and therapy monitoring of psoriasis inflammatory disease in vivo(Washington, DC : Optica, 2021-1-28) Schleusener, Johannes; Guo, Shuxia; Darvin, Maxim E.; Thiede, Gisela; Chernavskaia, Olga; Knorr, Florian; Lademann, Jürgen; Popp, Jürgen; Bocklitz, Thomas W.Psoriasis is considered a widespread dermatological disease that can strongly affect the quality of life. Currently, the treatment is continued until the skin surface appears clinically healed. However, lesions appearing normal may contain modifications in deeper layers. To terminate the treatment too early can highly increase the risk of relapses. Therefore, techniques are needed for a better knowledge of the treatment process, especially to detect the lesion modifications in deeper layers. In this study, we developed a fiber-based SORS-SERDS system in combination with machine learning algorithms to non-invasively determine the treatment efficiency of psoriasis. The system was designed to acquire Raman spectra from three different depths into the skin, which provide rich information about the skin modifications in deeper layers. This way, it is expected to prevent the occurrence of relapses in case of a too short treatment. The method was verified with a study of 24 patients upon their two visits: the data is acquired at the beginning of a standard treatment (visit 1) and four months afterwards (visit 2). A mean sensitivity of ≥85% was achieved to distinguish psoriasis from normal skin at visit 1. At visit 2, where the patients were healed according to the clinical appearance, the mean sensitivity was ≈65%.
- ItemActivity and electron donor preference of two denitrifying bacterial strains identified by Raman gas spectroscopy(Berlin [u.a.] : Springer, 2022) Blohm, Annika; Kumar, Swatantar; Knebl, Andreas; Herrmann, Martina; Küsel, Kirsten; Popp, Jürgen; Frosch, TorstenHuman activities have greatly increased the input of reactive nitrogen species into the environment and disturbed the balance of the global N cycle. This imbalance may be offset by bacterial denitrification, an important process in maintaining the ecological balance of nitrogen. However, our understanding of the activity of mixotrophic denitrifying bacteria is not complete, as most research has focused on heterotrophic denitrification. The aim of this study was to investigate substrate preferences for two mixotrophic denitrifying bacterial strains, Acidovorax delafieldii and Hydrogenophaga taeniospiralis, under heterotrophic, autotrophic or mixotrophic conditions. This complex analysis was achieved by simultaneous identification and quantification of H2, O2, CO2, 14N2, 15N2 and 15N2O in course of the denitrification process with help of cavity-enhanced Raman spectroscopic (CERS) multi-gas analysis. To disentangle electron donor preferences for both bacterial strains, microcosm-based incubation experiments under varying substrate conditions were conducted. We found that Acidovorax delafieldii preferentially performed heterotrophic denitrification in the mixotrophic sub-experiments, while Hydrogenophaga taeniospiralis preferred autotrophic denitrification in the mixotrophic incubation. These observations were supported by stoichiometric calculations. The results demonstrate the prowess of advanced Raman multi-gas analysis to study substrate use and electron donor preferences in denitrification, based on the comprehensive quantification of complex microbial gas exchange processes. © 2021, The Author(s).
- ItemIsolation of bacteria from artificial bronchoalveolar lavage fluid using density gradient centrifugation and their accessibility by Raman spectroscopy(Berlin [u.a.] : Springer, 2021) Wichmann, Christina; Rösch, Petra; Popp, JürgenRaman spectroscopy is an analytical method to identify medical samples of bacteria. Because Raman spectroscopy detects the biochemical properties of a cell, there are many factors that can influence and modify the Raman spectra of bacteria. One possible influence is a proper method for isolation of the bacteria. Medical samples in particular never occur in purified form, so a Raman-compatible isolation method is needed which does not affect the bacteria and thus the resulting spectra. In this study, we present a Raman-compatible method for isolation of bacteria from bronchoalveolar lavage (BAL) fluid using density gradient centrifugation. In addition to measuring the bacteria from a patient sample, the yield and the spectral influence of the isolation on the bacteria were investigated. Bacteria isolated from BAL fluid show additional peaks in comparison to pure culture bacteria, which can be attributed to components in the BAL sample. The isolation gradient itself has no effect on the spectra, and with a yield of 63% and 78%, the method is suitable for isolation of low concentrations of bacteria from a complex matrix. Graphical abstract.
- ItemMultimodal Molecular Imaging and Identification of Bacterial Toxins Causing Mushroom Soft Rot and Cavity Disease(Weinheim : Wiley-VCH, 2021) Dose, Benjamin; Thongkongkaew, Tawatchai; Zopf, David; Kim, Hak Joong; Bratovanov, Evgeni V.; García-Altares, María; Scherlach, Kirstin; Kumpfmüller, Jana; Ross, Claudia; Hermenau, Ron; Niehs, Sarah; Silge, Anja; Hniopek, Julian; Schmitt, Michael; Popp, Jürgen; Hertweck, ChristianSoft rot disease of edible mushrooms leads to rapid degeneration of fungal tissue and thus severely affects farming productivity worldwide. The bacterial mushroom pathogen Burkholderia gladioli pv. agaricicola has been identified as the cause. Yet, little is known about the molecular basis of the infection, the spatial distribution and the biological role of antifungal agents and toxins involved in this infectious disease. We combine genome mining, metabolic profiling, MALDI-Imaging and UV Raman spectroscopy, to detect, identify and visualize a complex of chemical mediators and toxins produced by the pathogen during the infection process, including toxoflavin, caryoynencin, and sinapigladioside. Furthermore, targeted gene knockouts and in vitro assays link antifungal agents to prevalent symptoms of soft rot, mushroom browning, and impaired mycelium growth. Comparisons of related pathogenic, mutualistic and environmental Burkholderia spp. indicate that the arsenal of antifungal agents may have paved the way for ancestral bacteria to colonize niches where frequent, antagonistic interactions with fungi occur. Our findings not only demonstrate the power of label-free, in vivo detection of polyyne virulence factors by Raman imaging, but may also inspire new approaches to disease control. © 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH
- ItemCorrelation of crystal violet biofilm test results of Staphylococcus aureus clinical isolates with Raman spectroscopic read-out(Chichester [u.a.] : Wiley, 2021) Ebert, Christina; Tuchscherr, Lorena; Unger, Nancy; Pöllath, Christine; Gladigau, Frederike; Popp, Jürgen; Löffler, Bettina; Neugebauer, UteBiofilm-related infections occur quite frequently in hospital settings and require rapid diagnostic identification as they are recalcitrant to antibiotic therapy and make special treatment necessary. One of the standard microbiological in vitro tests is the crystal violet test. It indirectly determines the amount of biofilm by measuring the optical density (OD) of the crystal violet-stained biofilm matrix and cells. However, this test is quite time-consuming, as it requires bacterial cultivation up to several days. In this study, we correlate fast Raman spectroscopic read-out of clinical Staphylococcus aureus isolates from 47 patients with different disease background with their biofilm-forming characteristics. Included were low (OD < 10), medium (OD ≥ 10 and ≤20), and high (OD > 20) biofilm performers as determined by the crystal violet test. Raman spectroscopic analysis of the bacteria revealed most spectral differences between high and low biofilm performers in the fingerprint region between 750 and 1150 cm−1. Using partial least square regression (PLSR) analysis on the Raman spectra involving the three categories of biofilm formation, it was possible to obtain a slight linear correlation of the Raman spectra with the biofilm OD values. The PLSR loading coefficient highlighted spectral differences between high and low biofilm performers for Raman bands that represent nucleic acids, carbohydrates, and proteins. Our results point to a possible application of Raman spectroscopy as a fast prediction tool for biofilm formation of bacterial strains directly after isolation from the infected patient. This could help clinicians make timely and adapted therapeutic decision in future.
- ItemRaman Stable Isotope Probing of Bacteria in Visible and Deep UV-Ranges(Basel : MDPI, 2021) Azemtsop Matanfack, Georgette; Pistiki, Aikaterini; Rösch, Petra; Popp, JürgenRaman stable isotope probing (Raman-SIP) is an excellent technique that can be used to access the overall metabolism of microorganisms. Recent studies have mainly used an excitation wavelength in the visible range to characterize isotopically labeled bacteria. In this work, we used UV resonance Raman spectroscopy (UVRR) to evaluate the spectral red-shifts caused by the uptake of isotopes (13C, 15N, 2H(D) and 18O) in E. coli cells. Moreover, we present a new approach based on the extraction of labeled DNA in combination with UVRR to identify metabolically active cells. The proof-of-principle study on E. coli revealed heterogeneities in the Raman features of both the bacterial cells and the extracted DNA after labeling with 13C, 15N, and D. The wavelength of choice for studying 18O- and deuterium-labeled cells is 532 nm is, while 13C-labeled cells can be investigated with visible and deep UV wavelengths. However, 15N-labeled cells are best studied at the excitation wavelength of 244 nm since nucleic acids are in resonance at this wavelength. These results highlight the potential of the presented approach to identify active bacterial cells. This work can serve as a basis for the development of new techniques for the rapid and efficient detection of active bacteria cells without the need for a cultivation step.
- ItemRaman imaging of changes in the polysaccharides distribution in the cell wall during apple fruit development and senescence(Berlin ; Heidelberg : Springer, 2016) Szymańska-Chargot, Monika; Chylińska, Monika; Pieczywek, Piotr M.; Rösch, Petra; Schmitt, Michael; Popp, Jürgen; Zdunek, ArturMain conclusion Du ring on-tree ripening, the pectin distribution changed from polydispersed in cell wall to cumulated in cell wall corners. During apple storage, the pectin distribution returned to evenly dispersed along the cell wall. The plant cell wall influences the texture properties of fruit tissue for example apples become softer during ripening and postharvest storage. This softening process is believed to be mainly connected with changes in the cell wall composition due to polysaccharides undergoing an enzymatic degradation. These changes in polysaccharides are currently mainly investigated via chemical analysis or monoclonal labeling. Here, we propose the application of Raman microscopy for evaluating the changes in the polysaccharide distribution in the cell wall of apples during both ripening and postharvest storage. The apples were harvested 1 month and 2 weeks before optimal harvest date as well as at the optimal harvest date. The apples harvested at optimal harvest date were stored for 3 months. The Raman maps, as well as the chemical analysis were obtained for each harvest date and after 1, 2 and 3 months of storage, respectively. The analysis of the Raman maps showed that the pectins in the middle lamella and primary cell wall undergo a degradation. The changes in cellulose and hemicellulose were less pronounced. These findings were confirmed by the chemical analysis results. During development changes of pectins from a polydispersed form in the cell walls to a cumulated form in cell wall corners could be observed. In contrast after 3 months of apple storage we could observe an substantial pectin decrease. The obtained results demonstrate that Raman chemical imaging might be a very useful tool for a first identification of compositional changes in plant tissue during their development. The great advantage Raman microspectroscopy offers is the simultaneous localization and identification of polysaccharides within the cell wall and plant tissue.