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Growth and metabolism of CHO-cells in porous glass carriers / Lüllau, E. ; Biselli, M. ; Wandrey, C.
(1994)
Several species of (poly)saccharides and organic acids can be found often simultaneously in various biological matrices, e.g., fruits, plant materials, and biological fluids. The analysis of such matrices sometimes represents a challenging task. Using Aloe vera (A. vera) plant materials as an example, the performance of several spectro-scopic methods (80 MHz benchtop NMR, NIR, ATR-FTIR and UV–vis) for the simultaneous analysis of quality parameters of this plant material was compared. The determined parameters include (poly)saccharides such as aloverose, fructose and glucose as well as organic acids (malic, lactic, citric, isocitric, acetic, fumaric, benzoic and sorbic acids). 500 MHz NMR and high-performance liquid chromatography (HPLC) were used as the reference methods.
UV–vis data can be used only for identification of added preservatives (benzoic and sorbic acids) and drying agent (maltodextrin) and semiquantitative analysis of malic acid. NIR and MIR spectroscopies combined with multivariate regression can deliver more informative overview of A. vera extracts being able to additionally quantify glucose, aloverose, citric, isocitric, malic, lactic acids and fructose. Low-field NMR measurements can be used for the quantification of aloverose, glucose, malic, lactic, acetic, and benzoic acids. The benchtop NMR method was successfully validated in terms of robustness, stability, precision, reproducibility and limit of detection (LOD) and quantification (LOQ), respectively. All spectroscopic techniques are useful for the screening of (poly)saccharides and organic acids in plant extracts and should be applied according to its availability as well as information and confidence required for the specific analytical goal. Benchtop NMR spectroscopy seems to be the most feasible solution for quality control of A. vera products.
Transcription-promoting genomic sites in mammalia: their elucidation and architectural principles
(1998)
The transgeneticist's toolbox: novel methods for the targeted modification of eukaryotic genomes
(2000)
Disruption experiments targeted at the Bacillus licheniformis degSU operon and GFP-reporter analysis provided evidence for promoter activity immediately upstream of degU. pMutin mediated concomitant introduction of the degU32 allele – known to cause hypersecretion in Bacillus subtilis – resulted in a marked increase in protease activity. Application of 5-fluorouracil based counterselection through establishment of a phosphoribosyltransferase deficient Δupp strain eventually facilitated the marker-free introduction of degU32 leading to further protease enhancement achieving levels as for hypersecreting wild strains in which degU was overexpressed. Surprisingly, deletion of rapG – known to interfere with DegU DNA-binding in B. subtilis – did not enhance protease production neither in the wild type nor in the degU32 strain. The combination of degU32 and Δupp counterselection in the type strain is not only equally effective as in hypersecreting wild strains with respect to protease production but furthermore facilitates genetic strain improvement aiming at biological containment and effectiveness of biotechnological processes.
A High-Throughput Functional Complementation Assay for Classification of BRCA1 Missense Variants
(2013)
[{ReN(PMe2Ph)3}{ReO3N}]2 – Structural Evidence for the Nitridotrioxorhenate(VII) Anion, [ReO3N]2−
(2005)
Poly(N-isopropylacrylamide) (PNIPAAm) hydrogel films with incorporated graphene oxide (GO) were developed and tested as light-stimulated actuators. GO dispersions were synthesized via Hummers method and characterized toward their optical properties and photothermal energy conversion. The hydrogels were prepared by means of photopolymerization. In addition, the influence of GO within the hydrogel network on the lower critical solution temperature (LCST) was investigated by differential scanning calorimetry (DSC). The optical absorbance and the response to illumination were determined as a function of GO concentration for thin hydrogel films. A proof of principle for the stimulation with light was performed.
Abstractauthoren Graphene oxide (GO) nanoparticles were incorporated in temperature-sensitive Poly(N-isopropylacrylamide) (PNIPAAm) hydrogels. The nanoparticles increase the light absorption and convert light energy into heat efficiently. Thus, the hydrogels with GO can be stimulated spatially resolved by illumination as it was demonstrated by IR thermography. The temporal progression of the temperature maximum was detected for different concentrations of GO within the polymer network. Furthermore, the compatibility of PNIPAAm hydrogels with GO and cell cultures was investigated. For this purpose, culture medium was incubated with hydrogels containing GO and the viability and morphology of chinese hamster ovary (CHO) cells was examined after several days of culturing in presence of this medium.
Lignin is a promising renewable biopolymer being investigated worldwide as an environmentally benign substitute of fossil-based aromatic compounds, e.g. for the use as an excipient with antioxidant and antimicrobial properties in drug delivery or even as active compound. For its successful implementation into process streams, a quick, easy, and reliable method is needed for its molecular weight determination. Here we present a method using 1H spectra of benchtop as well as conventional NMR systems in combination with multivariate data analysis, to determine lignin’s molecular weight (Mw and Mn) and polydispersity index (PDI). A set of 36 organosolv lignin samples (from Miscanthus x giganteus, Paulownia tomentosa and Silphium perfoliatum) was used for the calibration and cross validation, and 17 samples were used as external validation set. Validation errors between 5.6% and 12.9% were achieved for all parameters on all NMR devices (43, 60, 500 and 600 MHz). Surprisingly, no significant difference in the performance of the benchtop and high-field devices was found. This facilitates the application of this method for determining lignin’s molecular weight in an industrial environment because of the low maintenance expenditure, small footprint, ruggedness, and low cost of permanent magnet benchtop NMR systems.
The molecular weight properties of lignins are one of the key elements that need to be analyzed for a successful industrial application of these promising biopolymers. In this study, the use of 1H NMR as well as diffusion-ordered spectroscopy (DOSY NMR), combined with multivariate regression methods, was investigated for the determination of the molecular weight (Mw and Mn) and the polydispersity of organosolv lignins (n = 53, Miscanthus x giganteus, Paulownia tomentosa, and Silphium perfoliatum). The suitability of the models was demonstrated by cross validation (CV) as well as by an independent validation set of samples from different biomass origins (beech wood and wheat straw). CV errors of ca. 7–9 and 14–16% were achieved for all parameters with the models from the 1H NMR spectra and the DOSY NMR data, respectively. The prediction errors for the validation samples were in a similar range for the partial least squares model from the 1H NMR data and for a multiple linear regression using the DOSY NMR data. The results indicate the usefulness of NMR measurements combined with multivariate regression methods as a potential alternative to more time-consuming methods such as gel permeation chromatography.
The investigation of the possibility to determine various characteristics of powder heparin (n = 115) was carried out with infrared spectroscopy. The evaluation of heparin samples included several parameters such as purity grade, distributing company, animal source as well as heparin species (i.e. Na-heparin, Ca-heparin, and heparinoids). Multivariate analysis using principal component analysis (PCA), soft independent modelling of class analogy (SIMCA), and partial least squares – discriminant analysis (PLS-DA) were applied for the modelling of spectral data. Different pre-processing methods were applied to IR spectral data; multiplicative scatter correction (MSC) was chosen as the most relevant.
Obtained results were confirmed by nuclear magnetic resonance (NMR) spectroscopy. Good predictive ability of this approach demonstrates the potential of IR spectroscopy and chemometrics for screening of heparin quality. This approach, however, is designed as a screening tool and is not considered as a replacement for either of the methods required by USP and FDA.
A microfluidic chip integrating amperometric enzyme sensors for the detection of glucose, glutamate and glutamine in cell-culture fermentation processes has been developed. The enzymes glucose oxidase, glutamate oxidase and glutaminase were immobilized by means of cross-linking with glutaraldehyde on platinum thin-film electrodes integrated within a microfluidic channel. The biosensor chip was coupled to a flow-injection analysis system for electrochemical characterization of the sensors. The sensors have been characterized in terms of sensitivity, linear working range and detection limit. The sensitivity evaluated from the respective peak areas was 1.47, 3.68 and 0.28 μAs/mM for the glucose, glutamate and glutamine sensor, respectively. The calibration curves were linear up to a concentration of 20 mM glucose and glutamine and up to 10 mM for glutamate. The lower detection limit amounted to be 0.05 mM for the glucose and glutamate sensor, respectively, and 0.1 mM for the glutamine sensor. Experiments in cell-culture medium have demonstrated a good correlation between the glutamate, glutamine and glucose concentrations measured with the chip-based biosensors in a differential-mode and the commercially available instrumentation. The obtained results demonstrate the feasibility of the realized microfluidic biosensor chip for monitoring of bioprocesses.
Two types of microvalves based on temperature-responsive poly(N-isopropylacrylamide) (PNIPAAm) and pH-responsive poly(sodium acrylate) (PSA) hydrogel films have been developed and tested. The PNIPAAm and PSA hydrogel films were prepared by means of in situ photopolymerization directly inside the fluidic channel of a microfluidic chip fabricated by combining Si and SU-8 technologies. The swelling/shrinking properties and height changes of the PNIPAAm and PSA films inside the fluidic channel were studied at temperatures of deionized water from 14 to 36 °C and different pH values (pH 3–12) of Titrisol buffer, respectively. Additionally, in separate experiments, the lower critical solution temperature (LCST) of the PNIPAAm hydrogel was investigated by means of a differential scanning calorimetry (DSC) and a surface plasmon resonance (SPR) method. Mass-flow measurements have shown the feasibility of the prepared hydrogel films to work as an on-chip integrated temperature- or pH-responsive microvalve capable to switch the flow channel on/off.