Refine
Year of publication
Institute
- INB - Institut für Nano- und Biotechnologien (557) (remove)
Language
- English (557) (remove)
Document Type
- Article (503)
- Conference Proceeding (39)
- Part of a Book (8)
- Doctoral Thesis (3)
- Book (2)
- Other (2)
Keywords
A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3.
From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45 °C. Specific activity of StmPr2 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 17 ± 2 U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2.
Persistent infection with high-risk human papillomaviruses (hrHPV) can result in the formation of anogenital cancers. As hrHPV proteins E6 and E7 are required for cancer initiation and maintenance, they are ideal targets for immunotherapeutic interventions. Previously, we have described the development of DNA vaccines for the induction of HPV16 E6 and E7 specific T cell immunity. These vaccines consist of ‘gene-shuffled’ (SH) versions of HPV16 E6 and E7 that were fused to Tetanus Toxin Fragment C domain 1 (TTFC) and were named TTFC-E6SH and TTFC-E7SH. Gene-shuffling was performed to avoid the risk of inducing malignant transformation at the vaccination site. Here, we describe the preclinical safety evaluation of these candidate vaccines by analysis of their transforming capacity in vitro using established murine fibroblasts (NIH 3T3 cells) and primary human foreskin keratinocytes (HFKs). We demonstrate that neither ectopic expression of TTFC-E6SH and TTFC-E7SH alone or in combination enabled NIH 3T3 cells to form colonies in soft agar. In contrast, expression of HPV16 E6WT and E7WT alone or in combination resulted in effective transformation. Similarly, retroviral transduction of HFKs from three independent donors with both TTFC-E6SH and TTFC-E7SH alone or in combination did not show any signs of immortalization. In contrast, the combined expression of E6WT and E7WT induced immortalization in HFKs from all donors. Based on these results we consider it justified to proceed to clinical evaluation of DNA vaccines encoding TTFC-E6SH and TTFC-E7SH in patients with HPV16 associated (pre)malignancies.
Chelate stabilization of a titanium(IV)–salan alkoxide by ligand exchange with 2,6-pyridinedicarboxylic acid (dipic) resulted in heptacoordinate complex 3 which is not redox-active, stable on silica gel and has increased aqueous stability. 3 is highly toxic in HeLa S3 and Hep G2 and has enhanced antitumor efficacy in a mouse cervical-cancer model.
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.
Label-free electrical detection of consecutive deoxyribonucleic acid (DNA) hybridization/denaturation by means of an array of individually addressable field-effect-based nanoplate silicon-on-insulator (SOI) capacitors modified with gold nanoparticles (Au-NP) is investigated. The proposed device detects charge changes on Au-NP/DNA hybrids induced by the hybridization or denaturation event. DNA hybridization was performed in a high ionic-strength solution to provide a high hybridization efficiency. On the other hand, to reduce the screening of the DNA charge by counter ions and to achieve a high sensitivity, the sensor signal induced by the hybridization and denaturation events was measured in a low ionic-strength solution. High sensor signals of about 120, 90, and 80 mV were registered after the DNA hybridization, denaturation, and re-hybridization events, respectively. Fluorescence microscopy has been applied as reference method to verify the DNA immobilization, hybridization, and denaturation processes. An electrostatic charge-plane model for potential changes at the gate surface of a nanoplate field-effect sensor induced by the DNA hybridization has been developed taking into account both the Debye length and the distance of the DNA charge from the gate surface.
Real-time and reliable monitoring of the biogas process is crucial for a stable and efficient operation of biogas production in order to avoid digester breakdowns. The concentration of dissolved hydrogen (H₂) represents one of the key parameters for biogas process control. In this work, a one-chip integrated combined amperometric/field-effect sensor for monitoring the dissolved H₂ concentration has been developed for biogas applications. The combination of two different transducer principles might allow a more accurate and reliable measurement of dissolved H₂ as an early warning indicator of digester failures. The feasibility of the approach has been demonstrated by simultaneous amperometric/field-effect measurements of dissolved H₂ concentrations in electrolyte solutions. Both, the amperometric and the field-effect transducer show a linear response behaviour in the H₂ concentration range from 0.1 to 3% (v/v) with a slope of 198.4 ± 13.7 nA/% (v/v) and 14.9 ± 0.5 mV/% (v/v), respectively.
Field-effect capacitive electrolyte-insulator-semiconductor (EIS) sensors functionalised with citrate-capped gold nanoparticles (AuNP) have been used for the electrostatic detection of macromolecules by their intrinsic molecular charge. The EIS sensor detects the charge changes in the AuNP/macromolecule hybrids induced by the adsorption or binding events. A feasibility of the proposed detection scheme has been exemplary demonstrated by realising EIS sensors for the detection of poly-D-lysine molecules.
In vitro studies of the degradation kinetic of biopolymers are essential for the design and optimization of implantable biomedical devices. In the presented work, a field-effect capacitive sensor has been applied for the real-time and in situ monitoring of degradation processes of biopolymers for the first time. The polymer-covered field-effect sensor is, in principle, capable to detect any changes in bulk, surface and interface properties of the polymer induced by degradation processes. The feasibility of this approach has been experimentally proven by using the commercially available biomedical polymer poly(D,L-lactic acid) (PDLLA) as a model system. PDLLA films of different thicknesses were deposited on the Ta₂O₅-gate surface of the field-effect structure from a polymer solution by means of spin-coating method. The polymer-modified field-effect sensors have been characterized by means of capacitance–voltage and impedance-spectroscopy method. The degradation of the PDLLA was accelerated by changing the degradation medium from neutral (pH 7.2) to alkaline (pH 9) condition, resulting in drastic changes in the capacitance and impedance spectra of the polymer-modified field-effect sensor.
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.
Using a cell-based gas biosensor for investigation of adverse effects of acetone vapors in vitro
(2013)
We present a sensor concept based on copper(II)oxide (CuO) nanofibres for the detection of hydrogen peroxide (H2O2) vapour in the percent per volume (% v/v) range. The fibres were produced by using the electrospinning technique. To avoid water condensation in the pores, the fibres were initially modified by an exposure to H2S to get an enclosed surface. By a thermal treatment at 350 °C the fibres were oxidised back to CuO. Thereby, the visible pores disappear which was verified by SEM analysis. The fibres show a decrease of resistance with increasing H2O2 concentration which is due to the fact that hydrogen peroxide is an oxidising gas and CuO a p-type semiconductor. The sensor shows a change of resistance within the minute range to the exposure until the maximum concentration of 6.9% v/v H2O2. At operating temperatures below 450 °C the corresponding sensor response to a concentration of 4.1% v/v increases. The sensor shows a good reproducibility of the signal at different measurements. CuO seems to be a suitable candidate for the detection of H2O2 vapour at high concentrations.
Resistance behaviour of the sensor under exposure to H2O2 vapours between 2.3 and 6.9% v/v at an operating temperature of 450 °C.
In this work, a multi-sensor chip for the investigation of the sensing properties of different types of metal oxides towards hydrogen peroxide in the ppm range is presented. The fabrication process and physical characterization of the multi-sensor chip are described. Pure SnO2 and WO3 as well as Pd- and Pt-doped SnO2 films are characterized in terms of their sensitivity to H2O2. The sensing films have been prepared by drop-coating of water-dispensed nano-powders. A physical characterization, including scanning electron microscopy and X-ray diffraction analysis of the deposited metal-oxide films, was done. From the measurements in hydrogen peroxide atmosphere, it could be shown, that all of the tested metal oxide films are suitable for the detection of H2O2 in the ppm range. The highest sensitivity and reproducibility was achieved using Pt-doped SnO2.
Calibration plot of a SnO2, WO3, Pt-, and Pd-doped SnO2 gas sensor for H2O2 concentrations in the ppm range.
A wireless sensor system based on the industrial ZigBee standard for low-rate wireless networking was developed that enables real-time monitoring of gaseous H2O2 during the package sterilization in aseptic food processes. The sensor system consists of a remote unit connected to a calorimetric gas sensor, which was already established in former works, and an external base unit connected to a laptop computer. The remote unit was built up by an XBee radio frequency (RF) module for data communication and a programmable system-on-chip controller to read out the sensor signal and process the sensor data, whereas the base unit is a second XBee RF module. For the rapid H2O2 detection on various locations inside the package that has to be sterilized, a novel read-out strategy of the calorimetric gas sensor was established, wherein the sensor response is measured within the short sterilization time and correlated with the present H2O2 concentration. In an exemplary measurement application in an aseptic filling machinery, the suitability of the new, wireless sensor system was demonstrated, wherein the influence of the gas velocity on the H2O2 distribution inside a package was determined and verified with microbiological tests.
The chemical imaging sensor is a device to visualize the spatial distribution of chemical species based on the principle of LAPS (light-addressable potentiometric sensor), which is a field-effect chemical sensor based on semiconductor. In this study, the chemical imaging sensor has been applied to investigate the ion profile of laminar flows in a microfluidic channel. The chemical images (pH maps) were collected in a Y-shaped microfluidic channel while injecting HCl and NaCl solutions into two branches. From the chemical images, it was clearly observed that the injected solutions formed laminar flows in the channel. In addition, ion diffusion across the laminar flows was observed, and the diffusion coefficient could be derived by fitting the pH profiles to the Fick's equation.
Semiconductor-based chemical imaging sensors, like the light-addressable potentiometric sensor (LAPS) or the pH-imaging sensor based on a charge-coupled device (CCD), are becoming a powerful tool for label-free imaging of biological phenomena. We have proposed a polyion-based enzymatic membrane to develop an acetylcholine (ACh) imaging sensor for neural cell-activity observations. In this study, a CCD-type ACh-imaging sensor and a LAPS-type ACh-imaging sensor were fabricated and the prospect of both sensors was clarified by making a comparison of their basic characteristics.