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Keywords
C-terminal truncation of a metagenome-derived detergent protease for effective expression in E. coli
(2010)
Recently, a new alkaline protease named HP70 showing highest homology to extracellular serine proteases of Stenotrophomonas maltophilia and Xanthomonas campestris was found in the course of a metagenome screening for detergent proteases (Niehaus et al., submitted for publication). Attempts to efficiently express the enzyme in common expression hosts had failed. This study reports on the realization of overexpression in Escherichia coli after structural modification of HP70. Modelling of HP70 resulted in a two-domain structure, comprising the catalytic domain and a C-terminal domain which includes about 100 amino acids. On the basis of the modelled structure the enzyme was truncated by deletion of most of the C-terminal domain yielding HP70-C477.
This structural modification allowed effective expression of active enzyme using E. coli BL21-Gold as the host. Specific activity of HP70-C477 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 30 ± 5 U/mg compared to 8 ± 1 U/mg of the native enzyme. HP70-C477 was most active at 40 °C and pH 7–11; these conditions are prerequisite for a potential application as detergent enzyme. Determination of kinetic parameters at 40 °C and pH = 9.5 resulted in KM = 0.23 ± 0.01 mM and kcat = 167.5 ± 3.6 s⁻¹. MS-analysis of peptide fragments obtained from incubation of HP70 and HP70-C477 with insulin B indicated that the C-terminal domain influences the cleavage preferences of the enzyme. Washing experiments confirmed the high potential of HP70-C477 as detergent protease.
The chemical imaging sensor is a semiconductor-based chemical sensor that can visualize the two-dimensional distribution of specific ions or molecules in the solution. In this study, we developed a miniaturized chemical imaging sensor system with an OLED display panel as a light source that scans the sensor plate. In the proposed configuration, the display panel is placed directly below the sensor plate and illuminates the back surface. The measured area defined by illumination can be arbitrarily customized to fit the size and the shape of the sample to be measured. The waveform of the generated photocurrent, the currentvoltage characteristics and the pH sensitivity were investigated and pH imaging with this miniaturized system was demonstrated.
Realization of a calorimetric gas sensor on polyimide foil for applications in aseptic food industry
(2010)
A calorimetric gas sensor is presented for the monitoring of gas-phase H2O2 at elevated temperature during sterilization processes in aseptic food industry. The sensor consists of two temperature-sensitive thin-film resistances built up on a polyimide foil with a thickness of 25 μm, which are passivated with a layer of SU-8 photo resist and catalytically activated with manganese(IV) oxide. Instead of an active heating structure, the calorimetric sensor utilizes the elevated temperature of an evaporated H2O2 aerosol. In an experimental set-up, the sensor has shown a sensitivity of 4.78 °C/(%v/v) in a H2O2 concentration range of 0 to 10% v/v at an evaporation temperature of 240 ∘C. Furthermore, the sensor possesses the same, unchanged sensor signal even at varied evaporation temperatures of the gas stream. The sensor characterization demonstrates the suitability of the calorimetric gas sensor for monitoring the efficiency of sterilization processes.
In this contribution, we focus on the detection of toxic gases with living eukaryotic cells. A cell-based gas sensor system, able to measure the effects of direct exposure of gases to cells in real-time, was set up. Impedance data as well as oxygen consumption of Chinese hamster lung fibroblast cells (V79) were analysed upon exposure to carbon monoxide (CO). The CO (diluted in wet synthetic air) affects the cell respiration as indicated by an attenuated respiration signal after the CO exposure as well as an instant increase of the capacitive part of the impedance signal during the gas exposure.
Chalcogenide glass materials as membranes for potentiometric sensors for chemical analysis in solutions have been studied since more than 20 years. In this work, an electrolyte–insulator–semiconductor structure was combined with chalcogenide glass membranes prepared by means of the pulsed laser deposition technique. Depending on the membrane composition a selectivity to different ions (Cd2+ and Pb2+) is achieved. The different sensor membranes have been physically characterised using microscopy, ellipsometry, profilometry, atomic force microscopy (AFM), scanning electron microscopy (SEM) and Rutherford backscattering spectrometry (RBS). The electrochemical behaviour has been investigated via capacitance/voltage (C/V) and constant capacitance (ConCap) measurements and results in a Cd2+ sensitivity of 23.1 ± 0.6 mV per decade in a linear range from 7 × 10−6 to 10−2 mol/l and 24.4 ± 0.5 mV per decade in a linear range from 5 × 10−6 to 10−2 mol/l for Pb2+, respectively.
Chemical imaging systems allow the visualisation of the distribution of chemical species on the sensor surface. This work represents a new flexible approach of read out in a light-addressable potentiometric sensor (LAPS) with the help of a digital light processing (DLP) set-up. The DLP, known well for video projectors, consists of a mirror-array MEMS device which allows fast and flexible generation of light patterns. With the help of these light patterns the sensor surface of the LAPS device can be read out sequentially in a raster like scheme (scanning LAPS). The DLP approach has several advantages compared to conventional scanning LAPS set-ups, e.g., the spot size, the shape and the intensity of the light pointer can be changed easily and no mechanical movement is necessary, which reduces the size of the set-up and increases the stability and speed of measurement.
A new approach for a label-free electrical detection of DNA hybridization and denaturation using an array of individually addressable field-effect nanoplate SOI (silicon-on-insulator) capacitors functionalized with gold nanoparticles is presented. By using a constant-capacitance measuring setup in a differential mode, signal changes of ∼110 mV and ∼70 mV have been registered after the DNA hybridization and denaturation events, respectively.
The development of new interfaces for (bio-)chemical sensors requires comprehensive analyses and testing. The light-addressable potentiometric sensor (LAPS) can be used as a platform to investigate the sensitivity of a newly developed interface towards (bio-)chemical agents. LAPS measurements are spatially resolved by utilisation of focused light beams to define individual measurement spots. In this work, a new digitally modulated LAPS set-up based on an FPGA design will be introduced to increase the number of measurement spots, to shorten the measurement time and to improve the measurement accuracy.
Microfabrication, characterization and analytical application of a new thin-film silver microsensor
(2009)
Layer-by-Layer Assembly of Carbon Nanotubes Incorporated in Light-Addressable Potentiometric Sensors
(2009)
Optoelectronic Properties of Nanostructured Ensembles Controlled by Biomolecular Logic Systems
(2008)
Sensing charged macromolecules with nanocrystalline diamond-based field-effect capacitive sensors
(2008)
Heavy metal detection with semiconductor devices based on PLD-prepared chalcogenide glass thin films
(2007)
Handheld measurement device for field-effect sensor structures: Practical evaluation and limitations
(2007)
Novel organic membrane-based thin-film microsensors for the determination of heavy metal cations
(2006)
A first step towards the fabrication and electrochemical evaluation of thin-film microsensors based on organic PVC membranes for the determination of Hg(II), Cd(II), Pb(II) and Cu(II) ions in solutions has been realised. The membrane-coating mixture used in the preparation of this new type of microsensors is incorporating PVC as supporting matrix, o-nitrophenyloctylether (o-NPOE) as solvent mediator and a recently synthesized Hg[dimethylglyoxime(phene)]2+ and Bis-(4-hydroxyacetophenone)-ethylenediamine as electroactive materials for Hg(II) and Cd(II), respectively. A set of three commercialised ionophores for Cd(II), Pb(II) and Cu(II) has been also used for comparison. Thin-film microsensors based on these membranes showed a Nernstian response of slope (26-30 mV/dec.) for the respective tested cations. The potentiometric response characteristics (linear range, pH range, detection limit and response time) are comparable with those obtained by conventional membranes as well as coated wire electrodes prepared from the same membrane. The realisation of the new organic membrane-based thin-film microsensors overcomes the problem of an insufficient selectivity of solid-state-based thinfilm sensors.
An H2O2 sensor for the application in industrial sterilisation processes has been developed. Therefore, automated sterilisation equipment at laboratory scale has been constructed using parts from industrial sterilisation facilities. In addition, a software tool has been developed for the control of the sterilisation equipment at laboratory scale. First measurements with the developed sensor set-up as part of the sterilisation equipment have been performed and the sensor has been physically characterised by optical microscopy and SEM.
GaAs-based Gunn diodes with graded AlGaAs hot electron injector heterostructures have been developed under the special needs in automotive applications. The fabrication of the Gunn diode chips was based on total substrate removal and processing of integrated Au heat sinks. Especially, the thermal and RF behavior of the diodes have been analyzed by DC, impedance and S-parameter measurements. The electrical investigations have revealed the functionality of the hot electron injector. An optimized layer structure could fulfill the requirements in adaptive cruise control (ACC) systems at 77 GHz with typical output power between 50 and 90 mW.
A new and simple method for nanostructuring using conventional photolithography and layer expansion or pattern-size reduction technique is presented, which can further be applied for the fabrication of different nanostructures and nano-devices. The method is based on the conversion of a photolithographically patterned metal layer to a metal-oxide mask with improved pattern-size resolution using thermal oxidation. With this technique, the pattern size can be scaled down to several nanometer dimensions. The proposed method is experimentally demonstrated by preparing nanostructures with different configurations and layouts, like circles, rectangles, trapezoids, “fluidic-channel”-, “cantilever”- and meander-type structures.
Functional testing and characterisation of ISFETs on wafer level by means of a micro-droplet cell
(2006)
A wafer-level functionality testing and characterisation system for ISFETs (ionsensitive field-effect transistor) is realised by means of integration of a specifically designed capillary electrochemical micro-droplet cell into a commercial wafer prober-station. The developed system allows the identification and selection of “good” ISFETs at the earliest stage and to avoid expensive bonding, encapsulation and packaging processes for nonfunctioning ISFETs and thus, to decrease costs, which are wasted for bad dies. The developed system is also feasible for wafer-level characterisation of ISFETs in terms of sensitivity, hysteresis and response time. Additionally, the system might be also utilised for wafer-level testing of further electrochemical sensors.