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- ItemDetection of Pseudomonas aeruginosa Metabolite Pyocyanin in Water and Saliva by Employing the SERS Technique(Basel : MDPI, 2017) Zukovskaja, Olga; Jahn, Izabella-Jolan; Weber, Karina; Cialla-May, Dana; Popp, JürgenPyocyanin (PYO) is a metabolite specific for Pseudomonas aeruginosa. In the case of immunocompromised patients, it is currently considered a biomarker for life-threating Pseudomonas infections. In the frame of this study it is shown, that PYO can be detected in aqueous solution by employing surface-enhanced Raman spectroscopy (SERS) combined with a microfluidic platform. The achieved limit of detection is 0.5 μM. This is ~2 orders of magnitude below the concentration of PYO found in clinical samples. Furthermore, as proof of principle, the SERS detection of PYO in the saliva of three volunteers was also investigated. This body fluid can be collected in a non-invasive manner and is highly chemically complex, making the detection of the target molecule challenging. Nevertheless, PYO was successfully detected in two saliva samples down to 10 μM and in one sample at a concentration of 25 μM. This indicates that the molecules present in saliva do not inhibit the efficient adsorption of PYO on the surface of the employed SERS active substrates.
- 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.
- ItemEosinophils and Neutrophils-Molecular Differences Revealed by Spontaneous Raman, CARS and Fluorescence Microscopy(Basel : MDPI, 2020) Dorosz, Aleksandra; Grosicki, Marek; Dybas, Jakub; Matuszyk, Ewelina; Rodewald, Marko; Meyer, Tobias; Popp, Jürgen; Malek, Kamilla; Baranska, MalgorzataLeukocytes are a part of the immune system that plays an important role in the host's defense against viral, bacterial, and fungal infections. Among the human leukocytes, two granulocytes, neutrophils (Ne) and eosinophils (EOS) play an important role in the innate immune system. For that purpose, eosinophils and neutrophils contain specific granules containing protoporphyrin-type proteins such as eosinophil peroxidase (EPO) and myeloperoxidase (MPO), respectively, which contribute directly to their anti-infection activity. Since both proteins are structurally and functionally different, they could potentially be a marker of both cells' types. To prove this hypothesis, UV-Vis absorption spectroscopy and Raman imaging were applied to analyze EPO and MPO and their content in leukocytes isolated from the whole blood. Moreover, leukocytes can contain lipidic structures, called lipid bodies (LBs), which are linked to the regulation of immune responses and are considered to be a marker of cell inflammation. In this work, we showed how to determine the number of LBs in two types of granulocytes, EOS and Ne, using fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy. Spectroscopic differences of EPO and MPO can be used to identify these cells in blood samples, while the detection of LBs can indicate the cell inflammation process.
- ItemEvaluation of shifted excitation Raman difference spectroscopy and comparison to computational background correction methods applied to biochemical Raman spectra(Basel : MDPI, 2017) Cordero, Eliana; Korinth, Florian; Stiebing, Clara; Krafft, Christoph; Schie, Iwan W.; Popp, JürgenRaman spectroscopy provides label-free biochemical information from tissue samples without complicated sample preparation. The clinical capability of Raman spectroscopy has been demonstrated in a wide range of in vitro and in vivo applications. However, a challenge for in vivo applications is the simultaneous excitation of auto-fluorescence in the majority of tissues of interest, such as liver, bladder, brain, and others. Raman bands are then superimposed on a fluorescence background, which can be several orders of magnitude larger than the Raman signal. To eliminate the disturbing fluorescence background, several approaches are available. Among instrumentational methods shifted excitation Raman difference spectroscopy (SERDS) has been widely applied and studied. Similarly, computational techniques, for instance extended multiplicative scatter correction (EMSC), have also been employed to remove undesired background contributions. Here, we present a theoretical and experimental evaluation and comparison of fluorescence background removal approaches for Raman spectra based on SERDS and EMSC.
- ItemNoise Sources and Requirements for Confocal Raman Spectrometers in Biosensor Applications(Basel : MDPI, 2021) Jahn, Izabella J.; Grjasnow, Alexej; John, Henry; Weber, Karina; Popp, Jürgen; Hauswald, WalterRaman spectroscopy probes the biochemical composition of samples in a non-destructive, non-invasive and label-free fashion yielding specific information on a molecular level. Nevertheless, the Raman effect is very weak. The detection of all inelastically scattered photons with highest efficiency is therefore crucial as well as the identification of all noise sources present in the system. Here we provide a study for performance comparison and assessment of different spectrometers for confocal Raman spectroscopy in biosensor applications. A low-cost, home-built Raman spectrometer with a complementary metal-oxide-semiconductor (CMOS) camera, a middle price-class mini charge-coupled device (CCD) Raman spectrometer and a laboratory grade confocal Raman system with a deeply cooled CCD detector are compared. It is often overlooked that the sample itself is the most important “optical” component in a Raman spectrometer and its properties contribute most significantly to the signal-to-noise ratio. For this purpose, different representative samples: a crystalline silicon wafer, a polypropylene sample and E. coli bacteria were measured under similar conditions using the three confocal Raman spectrometers. We show that biosensor applications do not in every case profit from the most expensive equipment. Finally, a small Raman database of three different bacteria species is set up with the middle price-class mini CCD Raman spectrometer in order to demonstrate the potential of a compact setup for pathogen discrimination.
- ItemWide Field Spectral Imaging with Shifted Excitation Raman Difference Spectroscopy Using the Nod and Shuffle Technique(Basel : MDPI, 2020) Korinth, Florian; Schmälzlin, Elmar; Stiebing, Clara; Urrutia, Tanya; Micheva, Genoveva; Sandin, Christer; Müller, André; Maiwald, Martin; Sumpf, Bernd; Krafft, Christoph; Tränkle, Günther; Roth, Martin M; Popp, JürgenWide field Raman imaging using the integral field spectroscopy approach was used as a fast, one shot imaging method for the simultaneous collection of all spectra composing a Raman image. For the suppression of autofluorescence and background signals such as room light, shifted excitation Raman difference spectroscopy (SERDS) was applied to remove background artifacts in Raman spectra. To reduce acquisition times in wide field SERDS imaging, we adapted the nod and shuffle technique from astrophysics and implemented it into a wide field SERDS imaging setup. In our adapted version, the nod corresponds to the change in excitation wavelength, whereas the shuffle corresponds to the shifting of charges up and down on a Charge-Coupled Device (CCD) chip synchronous to the change in excitation wavelength. We coupled this improved wide field SERDS imaging setup to diode lasers with 784.4/785.5 and 457.7/458.9 nm excitation and applied it to samples such as paracetamol and aspirin tablets, polystyrene and polymethyl methacrylate beads, as well as pork meat using multiple accumulations with acquisition times in the range of 50 to 200 ms. The results tackle two main challenges of SERDS imaging: gradual photobleaching changes the autofluorescence background, and multiple readouts of CCD detector prolong the acquisition time.
- ItemApplication of High-Throughput Screening Raman Spectroscopy (HTS-RS) for Label-Free Identification and Molecular Characterization of Pollen(Basel : MDPI, 2019) Mondol, Abdullah S.; Patel, Milind D.; Rüger, Jan; Stiebing, Clara; Kleiber, Andreas; Henkel, Thomas; Popp, Jürgen; Schie, Iwan W.Pollen studies play a critical role in various fields of science. In the last couple of decades, replacement of manual identification of pollen by image-based methods using pollen morphological features was a great leap forward, but challenges for pollen with similar morphology remain, and additional approaches are required. Spectroscopy approaches for identification of pollen, such as Raman spectroscopy has potential benefits over traditional methods, due to the investigation of the intrinsic molecular composition of a sample. However, current Raman-based characterization of pollen is complex and time-consuming, resulting in low throughput and limiting the statistical significance of the acquired data. Previously demonstrated high-throughput screening Raman spectroscopy (HTS-RS) eliminates the complexity as well as human interaction by incorporation full automation of the data acquisition process. Here, we present a customization of HTS-RS for pollen identification, enabling sampling of a large number of pollen in comparison to other state-of-the-art Raman pollen investigations. We show that using Raman spectra we are able to provide a preliminary estimation of pollen types based on growth habits using hierarchical cluster analysis (HCA) as well as good taxonomy of 37 different Pollen using principal component analysis-support vector machine (PCA-SVM) with good accuracy even for the pollen specimens sharing similar morphological features. Our results suggest that HTS-RS platform meets the demands for automated pollen detection making it an alternative method for research concerning pollen.
- ItemRaman Signal Enhancement Tunable by Gold-Covered Porous Silicon Films with Different Morphology(Basel : MDPI, 2020) Agafilushkina, Svetlana N.; Žukovskaja, Olga; Dyakov, Sergey A.; Weber, Karina; Sivakov, Vladimir; Popp, Jürgen; Cialla-May, Dana; Osminkina, Liubov A.The ease of fabrication, large surface area, tunable pore size and morphology as well surface modification capabilities of a porous silicon (PSi) layer make it widely used for sensoric applications. The pore size of a PSi layer can be an important parameter when used as a matrix for creating surface-enhanced Raman scattering (SERS) surfaces. Here, we evaluated the SERS activity of PSi with pores ranging in size from meso to macro, the surface of which was coated with gold nanoparticles (Au NPs). We found that different pore diameters in the PSi layers provide different morphology of the gold coating, from an almost monolayer to 50 nm distance between nanoparticles. Methylene blue (MB) and 4-mercaptopyridine (4-MPy) were used to describe the SERS activity of obtained Au/PSi surfaces. The best Raman signal enhancement was shown when the internal diameter of torus-shaped Au NPs is around 35 nm. To understand the role of plasmonic resonances in the observed SERS spectrum, we performed electromagnetic simulations of Raman scattering intensity as a function of the internal diameter. The results of these simulations are consistent with the obtained experimental data
- ItemTopUp SERS substrates with integrated internal standard(Basel : MDPI, 2018) Patze, Sophie; Hübner, Uwe; Weber, Karina; Cialla-May, Dana; Popp, JürgenSurface-enhanced Raman spectroscopy (SERS) is known as a molecular-specific and highly sensitive method. In order to enable the routine application of SERS, powerful SERS substrates are of great importance. Within this manuscript, a TopUp SERS substrate is introduced which is fabricated by a top-down process based on microstructuring as well as a bottom-up generation of silver nanostructures. The Raman signal of the support material acts as an internal standard in order to improve the quantification capabilities. The analyte molecule coverage of sulfamethoxazole on the surface of the nanostructures is characterized by the SERS signal evolution fitted by a Langmuir–Freundlich isotherm.
- ItemFLIm and raman spectroscopy for investigating biochemical changes of bovine pericardium upon genipin cross-linking(Basel : MDPI, 2020) Shaik, Tanveer Ahmed; Alfonso-Garcia, Alba; Richter, Martin; Korinth, Florian; Krafft, Christoph; Marcu, Laura; Popp, JürgenBiomaterials used in tissue engineering and regenerative medicine applications benefit from longitudinal monitoring in a non-destructive manner. Label-free imaging based on fluorescence lifetime imaging (FLIm) and Raman spectroscopy were used to monitor the degree of genipin (GE) cross-linking of antigen-removed bovine pericardium (ARBP) at three incubation time points (0.5, 1.0, and 2.5 h). Fluorescence lifetime decreased and the emission spectrum redshifted compared to that of uncross-linked ARBP. The Raman signature of GE-ARBP was resonance-enhanced due to the GE cross-linker that generated new Raman bands at 1165, 1326, 1350, 1380, 1402, 1470, 1506, 1535, 1574, 1630, 1728, and 1741 cm-1. These were validated through density functional theory calculations as cross-linker-specific bands. A multivariate multiple regression model was developed to enhance the biochemical specificity of FLIm parameters fluorescence intensity ratio (R2 = 0.92) and lifetime (R2 = 0.94)) with Raman spectral results. FLIm and Raman spectroscopy detected biochemical changes occurring in the collagenous tissue during the cross-linking process that were characterized by the formation of a blue pigment which affected the tissue fluorescence and scattering properties. In conclusion, FLIm parameters and Raman spectroscopy were used to monitor the degree of cross-linking non-destructively. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.