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Institute
- INB - Institut für Nano- und Biotechnologien (613) (remove)
This work describes the novel combination of the light-addressable electrode (LAE) and the light-addressable potentiometric sensor (LAPS) into a microsystem set-up. Both the LAE as well as the LAPS shares the principle of addressing the active spot by means of a light beam. This enables both systems to manipulate resp. to detect an analyte with a high spatial resolution. Hence, combining both principles into a single set-up enables the active stimulation e.g., by means of electrolysis and a simultaneous observation e.g., the response of an entrapped biological cell by detection of extracellular pH changes. The work will describe the principles of both technologies and the necessary steps to integrate them into a single set-up. Furthermore, examples of application and operation of such systems will be presented.
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.
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.
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.
"Biologie trifft Mikroelektronik", das Motto des Instituts für Nano- und Biotechnologien (INB) an der FH Aachen, unterstreicht die zunehmende Bedeutung interdisziplinär geprägter Forschungsaktivitäten. Der thematische Zusammenschluss grundständiger Disziplinen, wie die Physik, Elektrotechnik, Chemie, Biologie sowie die Materialwissenschaften, lässt neue Forschungsgebiete entstehen, ein herausragendes Beispiel hierfür ist die Nanotechnologie: Hier werden neue Werkstoffe und Materialien entwickelt, einzelne Nanopartikel oder Moleküle und deren Wechselwirkung untersucht oder Schichtstrukturen im Nanometerbereich aufgebaut, die neue und vorher nicht bekannte Eigenschaften hervorbringen.
Vor diesem Hintergrund bündelt das im Jahre 2006 gegründete INB die an der FH Aachen vorhandenen Kompetenzen von derzeit insgesamt sieben Laboratorien auf den Gebieten der Halbleitertechnik und Nanoelektronik, Nanostrukturen und DNA-Sensorik, der Chemo- und Biosensorik, der Enzymtechnologie, der Mikrobiologie und Pflanzenbiotechnologie, der Zellkulturtechnik, sowie der Roten Biotechnologie synergetisch. In der Nano- und Biotechnologie steckt außergewöhnliches Potenzial! Nicht zuletzt deshalb stellen sich die Forscher der Herausforderung, in diesem Bereich gemeinsam zu forschen und Schnittstellen zu nutzen, um so bei der Gestaltung neuartiger Ideen und Produkte mitzuwirken, die zukünftig unser alltägliches Leben verändern werden.
Im Folgenden werden die verschiedenen Forschungsbereiche kurz zusammenfassend vorgestellt und vorhandene Interaktionen anhand von exemplarisch ausgewählten, aktuellen Forschungsprojekten skizziert.
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.
Die Detektion von Schadstoffen repräsentiert in der Umweltanalytik eine wichtige Aufgabenstellung. Gerade die Abwasser- bzw. Brauchwasseranalytik sowie die Prozesskontrolle haben einen hohen Stellenwert. Siliziumbasierte Dünnschichtsensoren bieten eine kostengünstige Möglichkeit, „online“-Messungen bzw. Vor-Ort-Messungen zeitnah durchzuführen. In dieser Arbeit wird ein potentiometrisches Sensorarray auf der Basis von Chalkogenidgläsern zur Detektion von Schwermetallen in wässrigen Medien vorgestellt.
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.
Using a cell-based gas biosensor for investigation of adverse effects of acetone vapors in vitro
(2013)
The response of Bacillus licheniformis to heat and ethanol stress and the role of the SigB regulon
(2013)
Size unlimited markerless deletions by a transconjugative plasmid-system in Bacillus licheniformis
(2013)
An application of a scanning light-addressable potentiometric sensor for label-free DNA detection
(2013)
Chemische Sensoren mit Bariumstrontiumtitanat als funktionelle Schicht zur Multiparameterdetektion
(2013)
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.
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.
The semiconductor field-effect platform represents a powerful tool for detecting the adsorption and binding of charged macromolecules with direct electrical readout. In this work, a capacitive electrolyte–insulator–semiconductor (EIS) field-effect sensor consisting of an Al-p-Si-SiO2 structure has been applied for real-time in situ electrical monitoring of the layer-by-layer formation of polyelectrolyte (PE) multilayers (PEM). The PEMs were deposited directly onto the SiO2 surface without any precursor layer or drying procedures. Anionic poly(sodium 4-styrene sulfonate) and cationic weak polyelectrolyte poly(allylamine hydrochloride) have been chosen as a model system. The effect of the ionic strength of the solution, polyelectrolyte concentration, number and polarity of the PE layers on the characteristics of the PEM-modified EIS sensors have been studied by means of capacitance–voltage and constant-capacitance methods. In addition, the thickness, surface morphology, roughness and wettabilityof the PE mono- and multilayers have been characterised by ellipsometry, atomic force microscopy and water contact-angle methods, respectively. To explain potential oscillations on the gate surface and signal behaviour of the capacitive field-effect EIS sensor modified with a PEM, a simplified electrostatic model that takes into account the reduced electrostatic screening of PE charges by mobile ions within the PEM has been proposed and discussed.
Living cells are complex biological systems transforming metabolites taken up from the surrounding medium. Monitoring the responses of such cells to certain substrate concentrations is a challenging task and offers possibilities to gain insight into the vitality of a community influenced by the growth environment. Cell-based sensors represent a promising platform for monitoring the metabolic activity and thus, the “welfare” of relevant organisms. In the present study, metabolic responses of the model bacterium Escherichia coli in suspension, layered onto a capacitive field-effect structure, were examined to pulses of glucose in the concentration range between 0.05 and 2 mM. It was found that acidification of the surrounding medium takes place immediately after glucose addition and follows Michaelis–Menten kinetic behavior as a function of the glucose concentration. In future, the presented setup can, therefore, be used to study substrate specificities on the enzymatic level and may as well be used to perform investigations of more complex metabolic responses. Conclusions and perspectives highlighting this system are discussed.
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.
In the present work, a novel method for monitoring sterilisation processes with gaseous H2O2 in combination with heat activation by means of a specially designed calorimetric gas sensor was evaluated. Therefore, the sterilisation process was extensively studied by using test specimens inoculated with Bacillus atrophaeus spores in order to identify the most influencing process factors on its microbicidal effectiveness. Besides the contact time of the test specimens with gaseous H2O2 varied between 0.2 and 0.5 s, the present H2O2 concentration in a range from 0 to 8% v/v (volume percent) had a strong influence on the microbicidal effectiveness, whereas the change of the vaporiser temperature, gas flow and humidity were almost negligible. Furthermore, a calorimetric H2O2 gas sensor was characterised in the sterilisation process with gaseous H2O2 in a wide range of parameter settings, wherein the measurement signal has shown a linear response against the H2O2 concentration with a sensitivity of 4.75 °C/(% v/v). In a final step, a correlation model by matching the measurement signal of the gas sensor with the microbial inactivation kinetics was established that demonstrates its suitability as an efficient method for validating the microbicidal effectiveness of sterilisation processes with gaseous H2O2.
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.