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- ItemA manual and an automatic TERS based virus discrimination(Cambridge : RSC Publ., 2015) Olschewski, Konstanze; Kämmer, Evelyn; Stöckel, Stephan; Bocklitz, Thomas; Deckert-Gaudig, Tanja; Zell, Roland; Cialla-May, Dana; Weber, Karina; Deckert, Volker; Popp, JürgenRapid techniques for virus identification are more relevant today than ever. Conventional virus detection and identification strategies generally rest upon various microbiological methods and genomic approaches, which are not suited for the analysis of single virus particles. In contrast, the highly sensitive spectroscopic technique tip-enhanced Raman spectroscopy (TERS) allows the characterisation of biological nano-structures like virions on a single-particle level. In this study, the feasibility of TERS in combination with chemometrics to discriminate two pathogenic viruses, Varicella-zoster virus (VZV) and Porcine teschovirus (PTV), was investigated. In a first step, chemometric methods transformed the spectral data in such a way that a rapid visual discrimination of the two examined viruses was enabled. In a further step, these methods were utilised to perform an automatic quality rating of the measured spectra. Spectra that passed this test were eventually used to calculate a classification model, through which a successful discrimination of the two viral species based on TERS spectra of single virus particles was also realised with a classification accuracy of 91%.
- ItemMolecular Specific and Sensitive Detection of Pyrazinamide and Its Metabolite Pyrazinoic Acid by Means of Surface Enhanced Raman Spectroscopy Employing In Situ Prepared Colloids(Basel : MDPI, 2019) Mühlig, Anna; Jahn, Izabella-Jolan; Heidler, Jan; Weber, Karina; Jahn, Martin; Sheen, Patricia; Zimic, Mirko; Cialla-May, Dana; Popp, JürgenThe prodrug pyrazinamide (PZA) is metabolized by the mycobacteria to pyrazinoic acid (POA), which is expelled into the extracellular environment. PZA resistance is highly associated to a lack of POA efflux. Thus, by detecting a reduction of the concentration of POA in the extracellular environment, by means of lab-on-a-chip (LoC)-SERS (surface-enhanced Raman spectroscopy), an alternative approach for the discrimination of PZA resistant mycobacteria is introduced. A droplet-based microfluidic SERS device has been employed to illustrate the potential of the LoC-SERS method for the discrimination of PZA resistant mycobacteria. The two analytes were detected discretely in aqueous solution with a limit of detection of 27 µm for PZA and 21 µm for POA. The simultaneous detection of PZA and POA in aqueous mixtures could be realized within a concentration range from 20 μm to 50 μm for PZA and from 50 μm to 80 μm for POA.
- 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.
- ItemIn Vitro Selection of Specific DNA Aptamers Against the Anti-Coagulant Dabigatran Etexilate(Berlin : Nature Publishing, 2018) Aljohani, Maher M; Chinnappan, Raja; Eissa, Shimaa; Alsager, Omar A; Weber, Karina; Cialla-May, Dana; Popp, Jürgen; Zourob, MohammedDabigatran Etexilate (PRADAXA) is a new oral anticoagulant increasingly used for a number of blood thrombosis conditions, prevention of strokes and systemic emboli among patients with atrial fibrillation. It provides safe and adequate anticoagulation for prevention and treatment of thrombus in several clinical settings. However, anticoagulation therapy can be associated with an increased risk of bleeding. There is a lack of specific laboratory tests to determine the level of this drug in blood. This is considered the most important obstacles of using this medication, particularly for patients with trauma, drug toxicity, in urgent need for surgical interventions or uncontrolled bleeding. In this work, we performed Systematic evolution of ligands by exponential enrichment (SELEX) to select specific DNA aptamers against dabigatran etexilate. Following multiple rounds of selection and enrichment with a randomized 60-mer DNA library, specific DNA aptamers for dabigatran were selected. We investigated the affinity and specificity of generated aptamers to the drug showing dissociation constants (Kd) ranging from 46.8–208 nM. The most sensitive aptamer sequence was selected and applied in an electrochemical biosensor to successfully achieve 0. 01 ng/ml level of detection of the target drug. With further improvement of the assay and optimization, these aptamers would replace conventional antibodies for developing detection assays in the near future.Dabigatran Etexilate (PRADAXA) is a new oral anticoagulant increasingly used for a number of blood thrombosis conditions, prevention of strokes and systemic emboli among patients with atrial fibrillation. It provides safe and adequate anticoagulation for prevention and treatment of thrombus in several clinical settings. However, anticoagulation therapy can be associated with an increased risk of bleeding. There is a lack of specific laboratory tests to determine the level of this drug in blood. This is considered the most important obstacles of using this medication, particularly for patients with trauma, drug toxicity, in urgent need for surgical interventions or uncontrolled bleeding. In this work, we performed Systematic evolution of ligands by exponential enrichment (SELEX) to select specific DNA aptamers against dabigatran etexilate. Following multiple rounds of selection and enrichment with a randomized 60-mer DNA library, specific DNA aptamers for dabigatran were selected. We investigated the affinity and specificity of generated aptamers to the drug showing dissociation constants (Kd) ranging from 46.8–208 nM. The most sensitive aptamer sequence was selected and applied in an electrochemical biosensor to successfully achieve 0. 01 ng/ml level of detection of the target drug. With further improvement of the assay and optimization, these aptamers would replace conventional antibodies for developing detection assays in the near future.
- 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.
- ItemLabel-free detection of Phytophthora ramorum using surface-enhanced Raman spectroscopy(Cambridge : Soc., 2015) Yüksel, Sezin; Schwenkbier, Lydia; Pollok, Sibyll; Weber, Karina; Cialla-May, Dana; Popp, JürgenIn this study, we report on a novel approach for the label-free and species-specific detection of the plant pathogen Phytophthora ramorum from real samples using surface enhanced Raman scattering (SERS). In this context, we consider the entire analysis chain including sample preparation, DNA isolation, amplification and hybridization on SERS substrate-immobilized adenine-free capture probes. Thus, the SERS-based detection of target DNA is verified by the strong spectral feature of adenine which indicates the presence of hybridized target DNA. This property was realized by replacing adenine moieties in the species-specific capture probes with 2-aminopurine. In the case of the matching capture and target sequence, the characteristic adenine peak serves as an indicator for specific DNA hybridization. Altogether, this is the first assay demonstrating the detection of a plant pathogen from an infected plant material by label-free SERS employing DNA hybridization on planar SERS substrates consisting of silver nanoparticles.
- ItemUV absorption spectroscopy in water-filled antiresonant hollow core fibers for pharmaceutical detection(Basel : MDPI, 2018) Nissen, Mona; Doherty, Brenda; Hamperl, J.; Kobelke, Jens; Weber, Karina; Henkel, Thomas; Schmidt, Markus A.Due to a worldwide increased use of pharmaceuticals and, in particular, antibiotics, a growing number of these substance residues now contaminate natural water resources and drinking supplies. This triggers a considerable demand for low-cost, high-sensitivity methods for monitoring water quality. Since many biological substances exhibit strong and characteristic absorption features at wavelengths shorter than 300 nm, UV spectroscopy presents a suitable approach for the quantitative identification of such water-contaminating species. However, current UV spectroscopic devices often show limited light-matter interaction lengths, demand sophisticated and bulky experimental infrastructure which is not compatible with microfluidics, and leave large fractions of the sample analyte unused. Here, we introduce the concept of UV spectroscopy in liquid-filled anti-resonant hollow core fibers, with large core diameters and lengths of approximately 1 m, as a means to overcome such limitations. This extended light-matter interaction length principally improves the concentration detection limit by two orders of magnitude while using almost the entire sample volume—that is three orders of magnitude smaller compared to cuvette based approaches. By integrating the fibers into an optofluidic chip environment and operating within the lowest experimentally feasible transmission band, concentrations of the application-relevant pharmaceutical substances, sulfamethoxazole (SMX) and sodium salicylate (SS), were detectable down to 0.1 µM (26 ppb) and 0.4 µM (64 ppb), respectively, with the potential to reach significantly lower detection limits for further device integration.
- 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.
- ItemRapid isolation and identification of pneumonia associated pathogens from sputum samples combining an innovative sample preparation strategy and array-based detection(Washington : American Chemical Society, 2019) Pahlow, Susanne; Lehniger, Lydia; Hentschel, Stefanie; Seise, Barbara; Braun, Sascha D.; Ehricht, Ralf; Berg, Albrecht; Popp, Jürgen; Weber, KarinaWith this study, an innovative and convenient enrichment and detection strategy for eight clinically relevant pneumonia pathogens, namely, Acinetobacter baumannii, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae is introduced. Bacteria were isolated from sputum samples with amine-modified particles exploiting pH-dependent electrostatic interactions between bacteria and the functionalized particle surface. Following this, an asymmetric polymerase chain reaction as well as subsequent stringent array-based hybridization with specific complementary capture probes were performed. Finally, results were visualized by an enzyme-induced silver nanoparticle deposition, providing stable endpoint signals and consequently an easy detection possibility. The assay was optimized using spiked samples of artificial sputum with different strains of the abovementioned bacterial species. Furthermore, actual patient sputum samples with S. pneumoniae were successfully analyzed. The presented approach offers great potential for the urgent need of a fast, specific, and reliable isolation and identification platform for important pneumonia pathogens, covering the complete process chain from sample preparation up to array-based detection within only 4 h.With this study, an innovative and convenient enrichment and detection strategy for eight clinically relevant pneumonia pathogens, namely, Acinetobacter baumannii, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae is introduced. Bacteria were isolated from sputum samples with amine-modified particles exploiting pH-dependent electrostatic interactions between bacteria and the functionalized particle surface. Following this, an asymmetric polymerase chain reaction as well as subsequent stringent array-based hybridization with specific complementary capture probes were performed. Finally, results were visualized by an enzyme-induced silver nanoparticle deposition, providing stable endpoint signals and consequently an easy detection possibility. The assay was optimized using spiked samples of artificial sputum with different strains of the abovementioned bacterial species. Furthermore, actual patient sputum samples with S. pneumoniae were successfully analyzed. The presented approach offers great potential for the urgent need of a fast, specific, and reliable isolation and identification platform for important pneumonia pathogens, covering the complete process chain from sample preparation up to array-based detection within only 4 h.
- 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.