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Keywords
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.
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.
A light-addressable potentiometric sensor (LAPS) can measure the concentration of one or several analytes at the sensor surface simultaneously in a spatially resolved manner. A modulated light pointer stimulates the semiconductor structure at the area of interest and a responding photocurrent can be read out. By simultaneous stimulation of several areas with light pointers of different modulation frequencies, the read out can be performed at the same time. With the new proposed controller electronic based on a field-programmable gate array (FPGA), it is possible to control the modulation frequencies, phase shifts, and light brightness of multiple light pointers independently and simultaneously. Thus, it is possible to investigate the frequency response of the sensor, and to examine the analyte concentration by the determination of the surface potential with the help of current/voltage curves and phase/voltage curves. Additionally, the ability to individually change the light intensities of each light pointer is used to perform signal correction.
Gas sensor investigation based on a catalytically activated thin-film thermopile for H2O2 detection
(2010)
Simultaneous detection of cyanide and heavy metals for environmental analysis by means of µISEs
(2010)
Bacillus subtilis and Bacillus licheniformis are widely used for the large-scale industrial production of proteins. These strains can efficiently secrete proteins into the culture medium using the general secretion (Sec) pathway. A characteristic feature of all secreted proteins is their N-terminal signal peptides, which are recognized by the secretion machinery. Here, we have studied the production of an industrially important secreted protease, namely, subtilisin BPN′ from Bacillus amyloliquefaciens. One hundred seventy-three signal peptides originating from B. subtilis and 220 signal peptides from the B. licheniformis type strain were fused to this secretion target and expressed in B. subtilis, and the resulting library was analyzed by high-throughput screening for extracellular proteolytic activity. We have identified a number of signal peptides originating from both organisms which produced significantly increased yield of the secreted protease. Interestingly, we observed that levels of extracellular protease were improved not only in B. subtilis, which was used as the screening host, but also in two different B. licheniformis strains. To date, it is impossible to predict which signal peptide will result in better secretion and thus an improved yield of a given extracellular target protein. Our data show that screening a library consisting of homologous and heterologous signal peptides fused to a target protein can identify more-effective signal peptides, resulting in improved protein export not only in the original screening host but also in different production strains.
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.
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.
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.
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)