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Keywords
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.
Control of Aharonov-Bohm oscillations in a AlGaAs/GaAs ring by asymmetric and symmetric gate biasing
(2001)
Aspergillus oryzae is an industrially relevant organism for the secretory production of heterologous enzymes, especially amylases. The activities of potential heterologous amylases, however, cannot be quantified directly from the supernatant due to the high background activity of native α-amylase. This activity is caused by the gene products of amyA, amyB, and amyC. In this study, an in vitro CRISPR/Cas9 system was established in A. oryzae to delete these genes simultaneously. First, pyrG of A. oryzae NSAR1 was mutated by exploiting NHEJ to generate a counter-selection marker. Next, all amylase genes were deleted simultaneously by co-transforming a repair template carrying pyrG of Aspergillus nidulans and flanking sequences of amylase gene loci. The rate of obtained triple knock-outs was 47%. We showed that triple knockouts do not retain any amylase activity in the supernatant. The established in vitro CRISPR/Cas9 system was used to achieve sequence-specific knock-in of target genes. The system was intended to incorporate a single copy of the gene of interest into the desired host for the development of screening methods. Therefore, an integration cassette for the heterologous Fpi amylase was designed to specifically target the amyB locus. The site-specific integration rate of the plasmid was 78%, with exceptional additional integrations. Integration frequency was assessed via qPCR and directly correlated with heterologous amylase activity. Hence, we could compare the efficiency between two different signal peptides. In summary, we present a strategy to exploit CRISPR/Cas9 for gene mutation, multiplex knock-out, and the targeted knock-in of an expression cassette in A. oryzae. Our system provides straightforward strain engineering and paves the way for development of fungal screening systems.
A concept for a new generation of an integrated multi-functional biosensor/actuator system is developed, which is based on biomolecular logic principles. Such a system is expected to be able to detect multiple biochemical input signals simultaneously and in real-time and convert them into electrical output signals with logical operations such as OR, AND, etc. The system can be designed as a closed-loop drug release device triggered by an enzyme logic gate, while the release of the drug induced by the actuator at the required dosage and timing will be controlled by an additional drug sensor. Thus, the system could help to make an accurate and specific diagnosis. The presented concept is exemplarily demonstrated by using an enzyme logic gate based on a glucose/glucose oxidase system, a temperature-responsive hydrogel mimicking the actuator function and an insulin (drug) sensor. In this work, the results of functional testing of individual amperometric glucose and insulin sensors as well as an impedimetric sensor for the detection of the hydrogel swelling/shrinking are presented.
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.
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.
A chip-based amperometric biosensor referring on using the bioelectrocatalytical amplification principle for the detection of low adrenaline concentrations is presented. The adrenaline biosensor has been prepared by modification of a platinum thin-film electrode with an enzyme membrane containing the pyrroloquinoline quinone-dependent glucose dehydrogenase and glutaraldehyde. Measuring conditions such as temperature, pH value, and glucose concentration have been optimized to achieve a high sensitivity and a low detection limit of about 1 nM adrenaline measured in phosphate buffer at neutral pH value. The response of the biosensor to different catecholamines has also been proven. Long-term stability of the adrenaline biosensor has been studied over 10 days. In addition, the biosensor has been successfully applied for adrenaline detection in human blood plasma for future biomedical applications. Furthermore, preliminary experiments have been carried to detect the adrenaline-concentration difference measured in peripheral blood and adrenal venous blood, representing the adrenal vein sampling procedure of a physician.
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.
Chemische Sensoren mit Bariumstrontiumtitanat als funktionelle Schicht zur Multiparameterdetektion
(2013)
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.
The chemical imaging sensor is a chemical sensor which is capable of visualizing the spatial distribution of chemical species in sample solution. In this study, a novel measurement system based on the chemical imaging sensor was developed to observe the inside of a Y-shaped microfluidic channel while injecting two sample solutions from two branches. From the collected chemical images, it was clearly observed that the injected solutions formed laminar flows in the microfluidic channel. In addition, ion diffusion across the laminar flows was observed. This label-free method can acquire quantitative data of ion distribution and diffusion in microfluidic devices, which can be used to determine the diffusion coefficients, and therefore, the molecular weights of chemical species in the sample solution.
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.
Characterising an insect antenna as a receptor for a biosensor by means of impedance spectroscopy
(2001)
Characterisation of polymeric materials as passivation layer for calorimetric H2O2 gas sensors
(2012)
Calorimetric gas sensors for monitoring the H₂O₂ concentration at elevated temperatures in industrial sterilisation processes have been presented in previous works. These sensors are built up in form of a differential set-up of a catalytically active and passive temperature-sensitive structure. Although, various types of catalytically active dispersions have been studied, the passivation layer has to be established and therefore, chemically as well as physically characterised. In the present work, fluorinated ethylene propylene (FEP), perfluoralkoxy (PFA) and epoxy-based SU-8 photoresist as temperature-stable polymeric materials have been investigated for sensor passivation in terms of their chemical inertness against H₂O₂, their hygroscopic properties as well as their morphology. The polymeric materials were deposited via spin-coating on the temperature-sensitive structure, wherein spin-coated FEP and PFA show slight agglomerates. However, they possess a low absorption of humidity due to their hydrophobic surface, whereas the SU-8 layer has a closed surface but shows a slightly higher absorption of water. All of them were inert against gaseous H₂O₂ during the characterisation in H₂O₂ atmosphere that demonstrates their suitability as passivation layer for calorimetric H₂O₂ gas sensors.
Background
Aminoacylases are highly promising enzymes for the green synthesis of acyl-amino acids, potentially replacing the environmentally harmful Schotten-Baumann reaction. Long-chain acyl-amino acids can serve as strong surfactants and emulsifiers, with application in cosmetic industries. Heterologous expression of these enzymes, however, is often hampered, limiting their use in industrial processes.
Results
We identified a novel mycobacterial aminoacylase gene from Mycolicibacterium smegmatis MKD 8, cloned and expressed it in Escherichia coli and Vibrio natriegens using the T7 overexpression system. The recombinant enzyme was prone to aggregate as inclusion bodies, and while V. natriegens Vmax™ could produce soluble aminoacylase upon induction with isopropyl β-d-1-thiogalactopyranoside (IPTG), E. coli BL21 (DE3) needed autoinduction with lactose to produce soluble recombinant protein. We successfully conducted a chaperone co-expression study in both organisms to further enhance aminoacylase production and found that overexpression of chaperones GroEL/S enhanced aminoacylase activity in the cell-free extract 1.8-fold in V. natriegens and E. coli. Eventually, E. coli ArcticExpress™ (DE3), which co-expresses cold-adapted chaperonins Cpn60/10 from Oleispira antarctica, cultivated at 12 °C, rendered the most suitable expression system for this aminoacylase and exhibited twice the aminoacylase activity in the cell-free extract compared to E. coli BL21 (DE3) with GroEL/S co-expression at 20 °C. The purified aminoacylase was characterized based on hydrolytic activities, being most stable and active at pH 7.0, with a maximum activity at 70 °C, and stability at 40 °C and pH 7.0 for 5 days. The aminoacylase strongly prefers short-chain acyl-amino acids with smaller, hydrophobic amino acid residues. Several long-chain amino acids were fairly accepted in hydrolysis as well, especially N-lauroyl-L-methionine. To initially evaluate the relevance of this aminoacylase for the synthesis of N-acyl-amino acids, we demonstrated that lauroyl-methionine can be synthesized from lauric acid and methionine in an aqueous system.
Conclusion
Our results suggest that the recombinant enzyme is well suited for synthesis reactions and will thus be further investigated.
Members of the species Bacillus pumilus get more and more in focus of the biotechnological industry as potential new production strains. Based on exoproteome analysis, B. pumilus strain Jo2, possessing a high secretion capability, was chosen for an omics-based investigation. The proteome and metabolome of B. pumilus cells growing either in minimal or complex medium was analyzed. In total, 1542 proteins were identified in growing B. pumilus cells, among them 1182 cytosolic proteins, 297 membrane and lipoproteins and 63 secreted proteins. This accounts for about 43% of the 3616 proteins encoded in the B. pumilus Jo2 genome sequence. By using GC–MS, IP-LC/MS and H NMR methods numerous metabolites were analyzed and assigned to reconstructed metabolic pathways. In the genome sequence a functional secretion system including the components of the Sec- and Tat-secretion machinery was found. Analysis of the exoproteome revealed secretion of about 70 proteins with predicted secretion signals. In addition, selected production-relevant genome features such as restriction modification systems and NRPS clusters of B. pumilus Jo2 are discussed.
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.
An ISFET-based penicillin sensor with high sensitivity, low detection limit and long lifetime
(2001)
An enzyme-based reversible Controlled NOT (CNOT) logic gate operating on a semiconductor transducer
(2017)
An enzyme-based biocatalytic system mimicking operation of a logically reversible Controlled NOT (CNOT) gate has been interfaced with semiconductor electronic transducers. Electrolyte–insulator–semiconductor (EIS) structures have been used to transduce chemical changes produced by the enzyme system to an electronically readable capacitive output signal using field-effect features of the EIS device. Two enzymes, urease and esterase, were immobilized on the insulating interface of EIS structure producing local pH changes performing XOR logic operation controlled by various combinations of the input signals represented by urea and ethyl butyrate. Another EIS transducer was functionalized with esterase only, thus performing Identity (ID) logic operation for the ethyl butyrate input. Both semiconductor devices assembled in parallel operated as a logically reversible CNOT gate. The present system, despite its simplicity, demonstrated for the first time logically reversible function of the enzyme system transduced electronically with the semiconductor devices. The biomolecular realization of a CNOT gate interfaced with semiconductors is promising for integration into complex biomolecular networks and future biosensor/biomedical applications.
An enzyme system organized in a flow device was used to mimic a reversible Controlled NOT (CNOT) gate with two input and two output signals. Reversible conversion of NAD⁺ and NADH cofactors was used to perform a XOR logic operation, while biocatalytic hydrolysis of p-nitrophenyl phosphate resulted in an Identity operation working in parallel. The first biomolecular realization of a CNOT gate is promising for integration into complex biomolecular networks and future biosensor/biomedical applications.
This work introduces a novel method for the detection of H₂O₂ vapor/aerosol of low concentrations, which is mainly applied in the sterilization of equipment in medical industry. Interdigitated electrode (IDE) structures have been fabricated by means of microfabrication techniques. A differential setup of IDEs was prepared, containing an active sensor element (active IDE) and a passive sensor element (passive IDE), where the former was immobilized with an enzymatic membrane of horseradish peroxidase that is selective towards H₂O₂. Changes in the IDEs’ capacitance values (active sensor element versus passive sensor element) under H₂O₂ vapor/aerosol atmosphere proved the detection in the concentration range up to 630 ppm with a fast response time (<60 s). The influence of relative humidity was also tested with regard to the sensor signal, showing no cross-sensitivity. The repeatability assessment of the IDE biosensors confirmed their stable capacitive signal in eight subsequent cycles of exposure to H₂O₂ vapor/aerosol. Room-temperature detection of H₂O₂ vapor/aerosol with such miniaturized biosensors will allow a future three-dimensional, flexible mapping of aseptic chambers and help to evaluate sterilization assurance in medical industry.
The on-chip integration of multiple biochemical sensors based on field-effect electrolyte-insulator-semiconductor capacitors (EISCAP) is challenging due to technological difficulties in realization of electrically isolated EISCAPs on the same Si chip. In this work, we present a new simple design for an array of on-chip integrated, individually electrically addressable EISCAPs with an additional control gate (CG-EISCAP). The existence of the CG enables an addressable activation or deactivation of on-chip integrated individual CG-EISCAPs by simple electrical switching the CG of each sensor in various setups, and makes the new design capable for multianalyte detection without cross-talk effects between the sensors in the array. The new designed CG-EISCAP chip was modelled in so-called floating/short-circuited and floating/capacitively-coupled setups, and the corresponding electrical equivalent circuits were developed. In addition, the capacitance-voltage curves of the CG-EISCAP chip in different setups were simulated and compared with that of a single EISCAP sensor. Moreover, the sensitivity of the CG-EISCAP chip to surface potential changes induced by biochemical reactions was simulated and an impact of different parameters, such as gate voltage, insulator thickness and doping concentration in Si, on the sensitivity has been discussed.
The characterization of the degradation kinetics of biodegradable polymers is mandatory with regard to their proper application. In the present work, polymer-modified electrolyte–insulator–semiconductor (PMEIS) field-effect sensors have been applied for in-situ monitoring of the pH-dependent degradation kinetics of the commercially available biopolymer poly(d,l-lactic acid) (PDLLA) in buffer solutions from pH 3 to pH 13. PDLLA films of 500 nm thickness were deposited on the surface of an Al–p-Si–SiO2–Ta2O5 structure from a polymer solution by means of spin-coating method. The PMEIS sensor is, in principle, capable to detect any changes in bulk, surface and interface properties of the polymer induced by degradation processes. A faster degradation has been observed for PDLLA films exposed to alkaline solutions (pH 9, pH 11 and pH 13).