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- 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-based evaluation of the membrane protein composition of the organohalide-respiring Sulfurospirillum multivorans(Chichester [u.a.] : Wiley, 2021) Cialla-May, Dana; Gadkari, Jennifer; Winterfeld, Andreea; Hübner, Uwe; Weber, Karina; Diekert, Gabriele; Schubert, Torsten; Goris, Tobias; Popp, JürgenBacteria often employ different respiratory chains that comprise membrane proteins equipped with various cofactors. Monitoring the protein inventory that is present in the cells under a given cultivation condition is often difficult and time-consuming. One example of a metabolically versatile bacterium is the microaerophilic organohalide-respiring Sulfurospirillum multivorans. Here, we used surface enhanced Raman spectroscopy (SERS) to quickly identify the cofactors involved in the respiration of S. multivorans. We cultured the organism with either tetrachloroethene (perchloroethylene, PCE), fumarate, nitrate, or oxygen as electron acceptors. Because the corresponding terminal reductases of the four different respiratory chains harbor different cofactors, specific fingerprint signals in SERS were expected. Silver nanostructures fabricated by means of electron beam lithography were coated with the membrane fractions extracted from the four S. multivorans cultivations, and SERS spectra were recorded. In the case of S. multivorans cultivated with PCE, the recorded SERS spectra were dominated by Raman peaks specific for Vitamin B12. This is attributed to the high abundance of the PCE reductive dehalogenase (PceA), the key enzyme in PCE respiration. After cultivation with oxygen, fumarate, or nitrate, no Raman spectral features of B12 were found. © 2020 The Authors. Journal of Raman Spectroscopy published by John Wiley & Sons Ltd
- 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.
- 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.
- ItemNon-instrumented DNA isolation, amplification and microarray-based hybridization for a rapid on-site detection of devastating Phytophthora kernoviae(Cambridge : Soc., 2015) Schwenkbier, Lydia; Pollok, Sibyll; Rudloff, Anne; Sailer, Sebastian; Cialla-May, Dana; Weber, Karina; Popp, JürgenA rapid and simple instrument-free detection system was developed for the identification of the plant pathogen Phytophthora kernoviae (P. kernoviae). The on-site operable analysis steps include magnetic particle based DNA isolation, helicase-dependent amplification (HDA) and chip-based DNA hybridization. The isothermal approach enabled the convenient amplification of the yeast GTP-binding protein (Ypt1) target gene in a miniaturized HDA-zeolite-heater (HZH) by an exothermic reaction. The amplicon detection on the chip was performed under room temperature conditions – either by successive hybridization and enzyme binding or by a combined step. A positive signal is displayed by enzymatically generated silver nanoparticle deposits, which serve as robust endpoint signals allowing an immediate visual readout. The hybridization assay enabled the reliable detection of 10 pg μL−1 target DNA. This is the first report of an entirely electricity-free, field applicable detection approach for devastating Phytophthora species, exemplarily shown for P. kernoviae.
- ItemToward food analytics: fast estimation of lycopene and β-carotene content in tomatoes based on surface enhanced Raman spectroscopy (SERS)(Cambridge : Soc., 2016) Radu, Andreea Ioana; Ryabchykov, Oleg; Bocklitz, Thomas Wilhelm; Huebner, Uwe; Weber, Karina; Cialla-May, Dana; Popp, JürgenCarotenoids are molecules that play important roles in both plant development and in the well-being of mammalian organisms. Therefore, various studies have been performed to characterize carotenoids’ properties, distribution in nature and their health benefits upon ingestion. Nevertheless, there is a gap regarding a fast detection of them at the plant phase. Within this contribution we report the results obtained regarding the application of surface enhanced Raman spectroscopy (SERS) toward the differentiation of two carotenoid molecules (namely, lycopene and β-carotene) in tomato samples. To this end, an e-beam lithography (EBL) SERS-active substrate and a 488 nm excitation source were employed, and a relevant simulated matrix was prepared (by mixing the two carotenoids in defined percentages) and measured. Next, carotenoids were extracted from tomato plants and measured as well. Finally, a combination of principal component analysis and partial least squares regression (PCA-PLSR) was applied to process the data, and the obtained results were compared with HPLC measurements of the same extracts. A good agreement was obtained between the HPLC and the SERS results for most of the tomato samples.