Refine
Year of publication
Institute
- INB - Institut für Nano- und Biotechnologien (615) (remove)
Language
- English (557)
- German (57)
- Multiple languages (1)
Document Type
- Article (542)
- Conference Proceeding (52)
- Part of a Book (9)
- Doctoral Thesis (4)
- Book (3)
- Other (2)
- Report (2)
- Patent (1)
Keywords
In this paper, carbon nanotubes (CNTs) were incorporated in penicillinase-phospholipid Langmuir and Langmuir–Blodgett (LB) films to enhance the enzyme catalytic properties. Adsorption of the penicillinase and CNTs at dimyristoylphosphatidic acid (DMPA) monolayers at the air–water interface was investigated by surface pressure–area isotherms, vibrational spectroscopy, and Brewster angle microscopy. The floating monolayers were transferred to solid supports through the LB technique, forming mixed DMPA-CNTs-PEN films, which were investigated by quartz crystal microbalance, vibrational spectroscopy, and atomic force microscopy. Enzyme activity was studied with UV–vis spectroscopy and the feasibility of the supramolecular device nanostructured as ultrathin films were essayed in a capacitive electrolyte–insulator–semiconductor (EIS) sensor device. The presence of CNTs in the enzyme–lipid LB film not only tuned the catalytic activity of penicillinase but also helped conserve its enzyme activity after weeks, showing increased values of activity. Viability as penicillin sensor was demonstrated with capacitance/voltage and constant capacitance measurements, exhibiting regular and distinctive output signals over all concentrations used in this work. These results may be related not only to the nanostructured system provided by the film, but also to the synergism between the compounds on the active layer, leading to a surface morphology that allowed a fast analyte diffusion because of an adequate molecular accommodation, which also preserved the penicillinase activity. This work therefore demonstrates the feasibility of employing LB films composed of lipids, CNTs, and enzymes as EIS devices for biosensing applications.
Algal polysaccharides (extracellular polysaccharides) and carbon nanotubes (CNTs) were adsorbed on dioctadecyldimethylammonium bromide Langmuir monolayers to serve as a matrix for the incorporation of urease. The physicochemical properties of the supramolecular system as a monolayer at the air–water interface were investigated by surface pressure–area isotherms, surface potential–area isotherms, interfacial shear rheology, vibrational spectroscopy, and Brewster angle microscopy. The floating monolayers were transferred to hydrophilic solid supports, quartz, mica, or capacitive electrolyte–insulator–semiconductor (EIS) devices, through the Langmuir–Blodgett (LB) technique, forming mixed films, which were investigated by quartz crystal microbalance, fluorescence spectroscopy, and field emission gun scanning electron microscopy. The enzyme activity was studied with UV–vis spectroscopy, and the feasibility of the thin film as a urea sensor was essayed in an EIS sensor device. The presence of CNT in the enzyme–lipid LB film not only tuned the catalytic activity of urease but also helped to conserve its enzyme activity. Viability as a urease sensor was demonstrated with capacitance–voltage and constant capacitance measurements, exhibiting regular and distinctive output signals over all concentrations used in this work. These results are related to the synergism between the compounds on the active layer, leading to a surface morphology that allowed fast analyte diffusion owing to an adequate molecular accommodation, which also preserved the urease activity. This work demonstrates the feasibility of employing LB films composed of lipids, CNT, algal polysaccharides, and enzymes as EIS devices for biosensing applications.
Electrolyte-insulator-semiconductor capacitors (EISCAP) belong to field-effect sensors having an attractive transducer architecture for constructing various biochemical sensors. In this study, a capacitive model of enzyme-modified EISCAPs has been developed and the impact of the surface coverage of immobilized enzymes on its capacitance-voltage and constant-capacitance characteristics was studied theoretically and experimentally. The used multicell arrangement enables a multiplexed electrochemical characterization of up to sixteen EISCAPs. Different enzyme coverages have been achieved by means of parallel electrical connection of bare and enzyme-covered single EISCAPs in diverse combinations. As predicted by the model, with increasing the enzyme coverage, both the shift of capacitance-voltage curves and the amplitude of the constant-capacitance signal increase, resulting in an enhancement of analyte sensitivity of the EISCAP biosensor. In addition, the capability of the multicell arrangement with multi-enzyme covered EISCAPs for sequentially detecting multianalytes (penicillin and urea) utilizing the enzymes penicillinase and urease has been experimentally demonstrated and discussed.
An acetoin biosensor based on a capacitive electrolyte–insulator–semiconductor (EIS) structure modified with the enzyme acetoin reductase, also known as butane-2,3-diol dehydrogenase (Bacillus clausii DSM 8716ᵀ), is applied for acetoin detection in beer, red wine, and fermentation broth samples for the first time. The EIS sensor consists of an Al/p-Si/SiO₂/Ta₂O₅ layer structure with immobilized acetoin reductase on top of the Ta₂O₅ transducer layer by means of crosslinking via glutaraldehyde. The unmodified and enzyme-modified sensors are electrochemically characterized by means of leakage current, capacitance–voltage, and constant capacitance methods, respectively.
Electrolyte-insulator-semiconductor (EIS) field-effect sensors belong to a new generation of electronic chips for biochemical sensing, enabling a direct electronic readout. The review gives an overview on recent advances and current trends in the research and development of chemical sensors and biosensors based on the capacitive field-effect EIS structure—the simplest field-effect device, which represents a biochemically sensitive capacitor. Fundamental concepts, physicochemical phenomena underlying the transduction mechanism and application of capacitive EIS sensors for the detection of pH, ion concentrations, and enzymatic reactions, as well as the label-free detection of charged molecules (nucleic acids, proteins, and polyelectrolytes) and nanoparticles, are presented and discussed.
Plant virus-like particles, and in particular, tobacco mosaic virus (TMV) particles, are increasingly being used in nano- and biotechnology as well as for biochemical sensing purposes as nanoscaffolds for the high-density immobilization of receptor molecules. The sensitive parameters of TMV-assisted biosensors depend, among others, on the density of adsorbed TMV particles on the sensor surface, which is affected by both the adsorption conditions and surface properties of the sensor. In this work, Ta₂O₅-gate field-effect capacitive sensors have been applied for the label-free electrical detection of TMV adsorption. The impact of the TMV concentration on both the sensor signal and the density of TMV particles adsorbed onto the Ta₂O₅-gate surface has been studied systematically by means of field-effect and scanning electron microscopy methods. In addition, the surface density of TMV particles loaded under different incubation times has been investigated. Finally, the field-effect sensor also demonstrates the label-free detection of penicillinase immobilization as model bioreceptor on TMV particles.
For the sterilisation of aseptic food packages it is taken advantage of the microbicidal properties of hydrogen peroxide (H2O2). Especially, when applied in vapour phase, it has shown high potential of microbial inactivation. In addition, it offers a high environmental compatibility compared to other chemical sterilisation agents, as it decomposes into oxygen and water, respectively. Due to a lack in sensory detection possibilities, a continuous monitoring of the H2O2 concentration was recently not available. Instead, the sterilisation efficacy is validated using microbiological tests. However, progresses in the development of calorimetric gas sensors during the last 7 years have made it possible to monitor the H2O2 concentration during operation. This chapter deals with the fundamentals of calorimetric gas sensing with special focus on the detection of gaseous hydrogen peroxide. A sensor principle based on a calorimetric differential set-up is described. Special emphasis is given to the sensor design with respect to the operational requirements under field conditions. The state-of-the-art regarding a sensor set-up for the on-line monitoring and secondly, a miniaturised sensor for in-line monitoring are summarised. Furthermore, alternative detection methods and a novel multi-sensor system for the characterisation of aseptic sterilisation processes are described.
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.
A new functionalization method to modify capacitive electrolyte–insulator–semiconductor (EIS) structures with nanofilms is presented. Layers of polyallylamine hydrochloride (PAH) and graphene oxide (GO) with the compound polyaniline:poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PANI:PAAMPSA) are deposited onto a p-Si/SiO2 chip using the layer-by-layer technique (LbL). Two different enzymes (urease and penicillinase) are separately immobilized on top of a five-bilayer stack of the PAH:GO/PANI:PAAMPSA-modified EIS chip, forming a biosensor for detection of urea and penicillin, respectively. Electrochemical characterization is performed by constant capacitance (ConCap) measurements, and the film morphology is characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). An increase in the average sensitivity of the modified biosensors (EIS–nanofilm–enzyme) of around 15% is found in relation to sensors, only carrying the enzyme but without the nanofilm (EIS–enzyme). In this sense, the nanofilm acts as a stable bioreceptor onto the EIS chip improving the output signal in terms of sensitivity and stability.
Halophilic and halotolerant microorganisms represent a promising source of salt-tolerant enzymes suitable for various biotechnological applications where high salt concentrations would otherwise limit enzymatic activity. Considering the current growing enzyme market and the need for more efficient and new biocatalysts, the present study aimed at the characterization of a high-alkaline subtilisin from Alkalihalobacillus okhensis Kh10-101T. The protease gene was cloned and expressed in Bacillus subtilis DB104. The recombinant protease SPAO with 269 amino acids belongs to the subfamily of high-alkaline subtilisins. The biochemical characteristics of purified SPAO were analyzed in comparison with subtilisin Carlsberg, Savinase, and BPN'. SPAO, a monomer with a molecular mass of 27.1 kDa, was active over a wide range of pH 6.0–12.0 and temperature 20–80 °C, optimally at pH 9.0–9.5 and 55 °C. The protease is highly oxidatively stable to hydrogen peroxide and retained 58% of residual activity when incubated at 10 °C with 5% (v/v) H2O2 for 1 h while stimulated at 1% (v/v) H2O2. Furthermore, SPAO was very stable and active at NaCl concentrations up to 5.0 m. This study demonstrates the potential of SPAO for biotechnological applications in the future.
Subtilisins from microbial sources, especially from the Bacillaceae family, are of particular interest for biotechnological applications and serve the currently growing enzyme market as efficient and novel biocatalysts. Biotechnological applications include use in detergents, cosmetics, leather processing, wastewater treatment and pharmaceuticals. To identify a possible candidate for the enzyme market, here we cloned the gene of the subtilisin SPFA from Fictibacillus arsenicus DSM 15822ᵀ (obtained through a data mining-based search) and expressed it in Bacillus subtilis DB104. After production and purification, the protease showed a molecular mass of 27.57 kDa and a pI of 5.8. SPFA displayed hydrolytic activity at a temperature optimum of 80 °C and a very broad pH optimum between 8.5 and 11.5, with high activity up to pH 12.5. SPFA displayed no NaCl dependence but a high NaCl tolerance, with decreasing activity up to concentrations of 5 m NaCl. The stability enhanced with increasing NaCl concentration. Based on its substrate preference for 10 synthetic peptide 4-nitroanilide substrates with three or four amino acids and its phylogenetic classification, SPFA can be assigned to the subgroup of true subtilisins. Moreover, SPFA exhibited high tolerance to 5% (w/v) SDS and 5% H₂O₂ (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H₂O₂, suggest it has potential for biotechnological applications.
Bestimmung der metabolischen Aktivität von Mikroorganismen während des Biogasbildungsprozesses
(2009)
Beryllium doped low-temperature-grown MBE GaAs: material for photomixing in the THz frequency range
(2000)
In this study, an online multi-sensing platform was engineered to simultaneously evaluate various process parameters of food package sterilization using gaseous hydrogen peroxide (H₂O₂). The platform enabled the validation of critical aseptic parameters. In parallel, one series of microbiological count reduction tests was performed using highly resistant spores of B. atrophaeus DSM 675 to act as the reference method for sterility validation. By means of the multi-sensing platform together with microbiological tests, we examined sterilization process parameters to define the most effective conditions with regards to the highest spore kill rate necessary for aseptic packaging. As these parameters are mutually associated, a correlation between different factors was elaborated. The resulting correlation indicated the need for specific conditions regarding the applied H₂O₂ gas temperature, the gas flow and concentration, the relative humidity and the exposure time. Finally, the novel multi-sensing platform together with the mobile electronic readout setup allowed for the online and on-site monitoring of the sterilization process, selecting the best conditions for sterility and, at the same time, reducing the use of the time-consuming and costly microbiological tests that are currently used in the food package industry.
Malaria infection remains a significant risk for much of the population of tropical and subtropical areas, particularly in developing countries. Therefore, it is of high importance to develop sensitive, accurate and inexpensive malaria diagnosis tests. Here, we present a novel aptamer-based electrochemical biosensor (aptasensor) for malaria detection by impedance spectroscopy, through the specific recognition between a highly discriminatory DNA aptamer and its target Plasmodium falciparum lactate dehydrogenase (PfLDH). Interestingly, due to the isoelectric point (pI) of PfLDH, the aptasensor response showed an adjustable detection range based on the different protein net-charge at variable pH environments. The specific aptamer recognition allows sensitive protein detection with an expanded detection range and a low detection limit, as well as a high specificity for PfLDH compared to analogous proteins. The specific feasibility of the aptasensor is further demonstrated by detection of the target PfLDH in human serum. Furthermore, the aptasensor can be easily regenerated and thus applied for multiple usages. The robustness, sensitivity, and reusability of the presented aptasensor make it a promising candidate for point-of-care diagnostic systems.
The chemical imaging sensor was applied to in-situ pH imaging of the solution in the vicinity of a corroding surface of stainless steel under potentiostatic polarization. A test piece of polished stainless steel was placed on the sensing surface leaving a narrow gap filled with artificial seawater and the stainless steel was corroded under polarization. The pH images obtained during polarization showed correspondence between the region of lower pH and the site of corrosion. It was also found that the pH value in the gap became as low as 2 by polarization, which triggered corrosion.
Multi-analyte biosensors may offer the opportunity to perform cost-effective and rapid analysis with reduced sample volume, as compared to electrochemical biosensing of each analyte individually. This work describes the development of an enzyme-based biosensor system for multi-parametric determination of four different organic acids. The biosensor array comprises five working electrodes for simultaneous sensing of ethanol, formate, d-lactate, and l-lactate, and an integrated counter electrode. Storage stability of the biosensor was evaluated under different conditions (stored at +4 °C in buffer solution and dry at −21 °C, +4 °C, and room temperature) over a period of 140 days. After repeated and regular application, the individual sensing electrodes exhibited the best stability when stored at −21 °C. Furthermore, measurements in silage samples (maize and sugarcane silage) were conducted with the portable biosensor system. Comparison with a conventional photometric technique demonstrated successful employment for rapid monitoring of complex media.
Application of a (bio-)chemical sensor (ISFET) for the detection of physical parameters in liquids
(2003)
Deoxyribonucleic acid (DNA) and protein recognition are now standard tools in biology. In addition, the special optical properties of microsphere resonators expressed by the high quality factor (Q-factor) of whispering gallery modes (WGMs) or morphology dependent resonances (MDRs) have attracted the attention of the biophotonic community. Microsphere-based biosensors are considered as powerful candidates to achieve label-free recognition of single molecules due to the high sensitivity of their WGMs. When the microsphere surface is modified with biomolecules, the effective refractive index and the effective size of the microsphere change resulting in a resonant wavelength shift. The transverse electric (TE) and the transverse magnetic (TM) elastic light scattering intensity of electromagnetic waves at 600 and 1400 nm are numerically calculated for DNA and unspecific binding of proteins to the microsphere surface. The effect of changing the optical properties was studied for diamond (refractive index 2.34), glass (refractive index 1.50), and sapphire (refractive index 1.75) microspheres with a 50 µm radius. The mode spacing, the linewidth of WGMs, and the shift of resonant wavelength due to the change in radius and refractive index, were analyzed by numerical simulations. Preliminary results of unspecific binding of biomolecules are presented. The calculated shift in WGMs can be used for biomolecules detection.