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- frequency mixing magnetic detection (4)
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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.
A light-addressable potentiometric sensor (LAPS) is a field-effect-based potentiometric device, which detects concentration changes of an analyte solution on the sensor surface in a spatially resolved way. It uses a light source to generate electron–hole pairs inside the semiconductor, which are separated in the depletion region due to an applied bias voltage across the sensor structure and hence, a surface-potential-dependent photocurrent can be read out. However, depending on the beam angle of the light source, scattering effects can occur, which influence the recorded signal in LAPS-based differential measurements. To solve this problem, a novel illumination unit based on a field programmable gate array (FPGA) consisting of 16 small-sized tunable infrared laser-diode modules (LDMs) is developed. Due to the improved focus of the LDMs with a beam angle of only 2 mrad, undesirable scattering effects are minimized. Escherichia coli (E. coli) K12 bacteria are used as a test microorganism to study the extracellular acidification on the sensor surface. Furthermore, a salt bridge chamber is built up and integrated with the LAPS system enabling multi-chamber differential measurements with a single Ag/AgCl reference electrode.
In this study, flexible calorimetric gas sensors are developed for specificdetection of gaseous hydrogen peroxide (H₂O₂) over a wide concentrationrange, which is used in sterilization processes for aseptic packaging industry.The flexibility of these sensors is an advantage for identifying the chemical components of the sterilant on the corners of the food boxes, so-called “coldspots”, as critical locations in aseptic packaging, which are of great importance. These sensors are fabricated on flexible polyimide films by means of thin-film technique. Thin layers of titanium and platinum have been deposited on polyimide to define the conductive structures of the sensors. To detect the high-temperature evaporated H₂O₂, a differential temperature set-up is proposed. The sensors are evaluated in a laboratory-scaled sterilizationsystem to simulate the sterilization process. The concentration range of the evaporated H₂O₂ from 0 to 7.7% v/v was defined and the sensors have successfully detected high as well as low H₂O₂ concentrations with a sensitivity of 5.04 °C/% v/v. The characterizations of the sensors confirm their precise fabrication, high sensitivity and the novelty of low H₂O₂ concentration detections for future inline monitoring of food-package sterilization.
In this work, a spore-based biosensor is evaluated to monitor the microbicidal efficacy of sterilization processes applying gaseous hydrogen peroxide (H2O2). The sensor is based on interdigitated electrode structures (IDEs) that have been fabricated by means of thin-film technologies. Impedimetric measurements are applied to study the effect of sterilization process on spores of Bacillus atrophaeus. This resilient microorganism is commonly used in industry to proof the sterilization efficiency. The sensor measurements are accompanied by conventional microbiological challenge tests, as well as morphological characterizations with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The sensor measurements are correlated with the microbiological test routines. In both methods, namely the sensor-based and microbiological one, a tailing effect has been observed. The results are evaluated and discussed in a three-dimensional calibration plot demonstrating the sensor's suitability to enable a rapid process decision in terms of a successfully performed sterilization.
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 is a semiconductor-based chemical sensor capable of visualizing pH and ion distributions. The spatial resolution depends on the lateral diffusion of photocarriers generated by illumination of the semiconductor substrate. In this study, two types of optical setups, one based on a bundle of optical fibers and the other based on a binocular tube head, were developed to project a hybrid illumination of a modulated light beam and a ring-shaped constant illumination onto the sensor plate. An improved spatial resolution was realized by the ring-shaped constant illumination, which suppressed lateral diffusion of photocarriers by enhanced recombination due to the increased carrier concentration.
Monitoring of organic acids (OA) and volatile fatty acids (VFA) is crucial for the control of anaerobic digestion. In case of unstable process conditions, an accumulation of these intermediates occurs. In the present work, two different enzyme-based biosensor arrays are combined and presented for facile electrochemical determination of several process-relevant analytes. Each biosensor utilizes a platinum sensor chip (14 × 14 mm²) with five individual working electrodes. The OA biosensor enables simultaneous measurement of ethanol, formate, d- and l-lactate, based on a bi-enzymatic detection principle. The second VFA biosensor provides an amperometric platform for quantification of acetate and propionate, mediated by oxidation of hydrogen peroxide. The cross-sensitivity of both biosensors toward potential interferents, typically present in fermentation samples, was investigated. The potential for practical application in complex media was successfully demonstrated in spiked sludge samples collected from three different biogas plants. Thereby, the results obtained by both of the biosensors were in good agreement to the applied reference measurements by photometry and gas chromatography, respectively. The proposed hybrid biosensor system was also used for long-term monitoring of a lab-scale biogas reactor (0.01 m³) for a period of 2 months. In combination with typically monitored parameters, such as gas quality, pH and FOS/TAC (volatile organic acids/total anorganic carbonate), the amperometric measurements of OA and VFA concentration could enhance the understanding of ongoing fermentation processes.
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.
A capacitive electrolyte-insulator-semiconductor (EIS) field-effect biosensor for acetoin detection has been presented for the first time. The EIS sensor consists of a layer structure of Al/p-Si/SiO₂/Ta₂O₅/enzyme acetoin reductase. The enzyme, also referred to as butane-2,3-diol dehydrogenase from B. clausii DSM 8716T, has been recently characterized. The enzyme catalyzes the (R)-specific reduction of racemic acetoin to (R,R)- and meso-butane-2,3-diol, respectively. Two different enzyme immobilization strategies (cross-linking by using glutaraldehyde and adsorption) have been studied. Typical biosensor parameters such as optimal pH working range, sensitivity, hysteresis, linear concentration range and long-term stability have been examined by means of constant-capacitance (ConCap) mode measurements. Furthermore, preliminary experiments have been successfully carried out for the detection of acetoin in diluted white wine samples.
An amperometric bi-enzyme biosensor based on substrate recycling principle for the amplification of the sensor signal has been developed for the detection of adrenaline in blood. Adrenaline can be used as biomarker verifying successful adrenal venous sampling procedure. The adrenaline biosensor has been realized via modification of a galvanic oxygen sensor with a bi-enzyme membrane combining a genetically modified laccase and a pyrroloquinoline quinone-dependent glucose dehydrogenase. The measurement conditions such as pH value and temperature were optimized to enhance the sensor performance. A high sensitivity and a low detection limit of about 0.5–1 nM adrenaline have been achieved in phosphate buffer at pH 7.4, relevant for measurements in blood samples. The sensitivity of the biosensor to other catecholamines such as noradrenaline, dopamine and dobutamine has been studied. Finally, the sensor has been successfully applied for the detection of adrenaline in human blood plasma.
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.
The presentation of enzymes on viral scaffolds has beneficial effects such as an increased enzyme loading and a prolonged reusability in comparison to conventional immobilization platforms. Here, we used modified tobacco mosaic virus (TMV) nanorods as enzyme carriers in penicillin G detection for the first time. Penicillinase enzymes were conjugated with streptavidin and coupled to TMV rods by use of a bifunctional biotin-linker. Penicillinase-decorated TMV particles were characterized extensively in halochromic dye-based biosensing. Acidometric analyte detection was performed with bromcresol purple as pH indicator and spectrophotometry. The TMV-assisted sensors exhibited increased enzyme loading and strongly improved reusability, and higher analysis rates compared to layouts without viral adapters. They extended the half-life of the sensors from 4 - 6 days to 5 weeks and thus allowed an at least 8-fold longer use of the sensors. Using a commercial budget-priced penicillinase preparation, a detection limit of 100 µM penicillin was obtained. Initial experiments also indicate that the system may be transferred to label-free detection layouts.
Field-effect-based electrolyte-insulator-semiconductor (EIS) sensors were modified with a bilayer of positively charged weak polyelectrolyte (poly(allylamine hydrochloride) (PAH)) and probe single-stranded DNA (ssDNA) and are used for the detection of complementary single-stranded target DNA (cDNA) in different test solutions. The sensing mechanism is based on the detection of the intrinsic molecular charge of target cDNA molecules after the hybridization event between cDNA and immobilized probe ssDNA. The test solutions contain synthetic cDNA oligonucleotides (with a sequence of tuberculosis mycobacteria genome) or PCR-amplified DNA (which origins from a template DNA strand that has been extracted from Mycobacterium avium paratuberculosis-spiked human sputum samples), respectively. Sensor responses up to 41 mV have been measured for the test solutions with DNA, while only small signals of ∼5 mV were detected for solutions without DNA. The lower detection limit of the EIS sensors was ∼0.3 nM, and the sensitivity was ∼7.2 mV/decade. Fluorescence experiments using SybrGreen I fluorescence dye support the electrochemical results.
In this work, a cell-based biosensor to evaluate the sterilization efficacy of hydrogen peroxide vapor sterilization processes is characterized. The transducer of the biosensor is based on interdigitated gold electrodes fabricated on an inert glass substrate. Impedance spectroscopy is applied to evaluate the sensor behavior and the alteration of test microorganisms due to the sterilization process. These alterations are related to changes in relative permittivity and electrical conductivity of the bacterial spores. Sensor measurements are conducted with and without bacterial spores (Bacillus atrophaeus), as well as after an industrial sterilization protocol. Equivalent two-dimensional numerical models based on finite element method of the periodic finger structures of the interdigitated gold electrodes are designed and validated using COMSOL® Multiphysics software by the application of known dielectric properties. The validated models are used to compute the electrical properties at different sensor states (blank, loaded with spores, and after sterilization). As a final result, we will derive and tabulate the frequency-dependent electrical parameters of the spore layer using a novel model that combines experimental data with numerical optimization techniques.
Magnetic detection structure for Lab-on-Chip applications based on the frequency mixing technique
(2018)
A magnetic frequency mixing technique with a set of miniaturized planar coils was investigated for use with a completely integrated Lab-on-Chip (LoC) pathogen sensing system. The system allows the detection and quantification of superparamagnetic beads. Additionally, in terms of magnetic nanoparticle characterization ability, the system can be used for immunoassays using the beads as markers. Analytical calculations and simulations for both excitation and pick-up coils are presented; the goal was to investigate the miniaturization of simple and cost-effective planar spiral coils. Following these calculations, a Printed Circuit Board (PCB) prototype was designed, manufactured, and tested for limit of detection, linear response, and validation of theoretical concepts. Using the magnetic frequency mixing technique, a limit of detection of 15 µg/mL of 20 nm core-sized nanoparticles was achieved without any shielding.
A field-effect biosensor employing tobacco mosaic virus (TMV) particles as scaffolds for enzyme immobilization is presented. Nanotubular TMV scaffolds allow a dense immobilization of precisely positioned enzymes with retained activity. To demonstrate feasibility of this new strategy, a penicillin sensor has been developed by coupling a penicillinase with virus particles as a model system. The developed field-effect penicillin biosensor consists of an Al-p-Si-SiO₂-Ta₂O₅-TMV structure and has been electrochemically characterized in buffer solutions containing different concentrations of penicillin G. In addition, the morphology of the biosensor surface with virus particles was characterized by scanning electron microscopy and atomic force microscopy methods. The sensors possessed a high penicillin sensitivity of ~ 92 mV/dec in a nearly-linear range from 0.1 mM to 10 mM, and a low detection limit of about 50 µM. The long-term stability of the penicillin biosensor was periodically tested over a time period of about one year without any significant loss of sensitivity. The biosensor has also been successfully applied for penicillin detection in bovine milk samples.
Cupriavidus necator H16 gains increasing attention in microbial research and biotechnological application due to its diverse metabolic features. Here we present a tightly controlled gene expression system for C. necator including the pBBR1-vector that contains hybrid promoters originating from C. necator native tolC-promoter in combination with a synthetic tetO-operator. The expression of the reporter gene from these plasmids relies on the addition of the exogenous inducer doxycycline (dc). The novel expression system offers a combination of advantageous features as; (i) high and dose-dependent recombinant protein production, (ii) tight control with a high dynamic range (On/Off ratio), which makes it applicable for harmful pathways or for toxic protein production, (iii) comparable cheap inducer (doxycycline, dc), (iv) effective at low inducer concentration, that makes it useful for large scale application, (v) rapid, diffusion controlled induction, and (vi) the inducer does not interfere within the cell metabolism. As applications of the expression system in C. necator H16, the growth ability on glycerol was enhanced by constitutively expressing the E. coli glpk gene-encoding for glycerol kinase. Likewise, we used the system to overcome the expression toxicity of mevalonate pathway in C. necator H16. With this system, the mevalonate-genes were successfully introduced in the host and the recombinant strains could produce about 200 mg/l mevalonate.
Monitoring the cellular metabolism of bacteria in (bio)fermentation processes is crucial to control and steer them, and to prevent undesired disturbances linked to metabolically inactive microorganisms. In this context, cell-based biosensors can play an important role to improve the quality and increase the yield of such processes. This work describes the simultaneous analysis of the metabolic behavior of three different types of bacteria by means of a differential light-addressable potentiometric sensor (LAPS) set-up. The study includes Lactobacillus brevis, Corynebacterium glutamicum, and Escherichia coli, which are often applied in fermentation processes in bioreactors. Differential measurements were carried out to compensate undesirable influences such as sensor signal drift, and pH value variation during the measurements. Furthermore, calibration curves of the cellular metabolism were established as a function of the glucose concentration or cell number variation with all three model microorganisms. In this context, simultaneous (bio)sensing with the multi-organism LAPS-based set-up can open new possibilities for a cost-effective, rapid detection of the extracellular acidification of bacteria on a single sensor chip. It can be applied to evaluate the metabolic response of bacteria populations in a (bio)fermentation process, for instance, in the biogas fermentation process.
Enzyme-catalyzed reactions have been designed to mimic various Boolean logic gates in the general framework of unconventional biomolecular computing. While some of the logic gates, particularly OR, AND, are easy to realize with biocatalytic reactions and have been reported in numerous publications, some other, like NXOR, are very challenging and have not been realized yet with enzyme reactions. The paper reports on a novel approach to mimicking the NXOR logic gate using the bell-shaped enzyme activity dependent on pH values. Shifting pH from the optimum value to the acidic or basic values by using acid or base inputs (meaning 1,0 and 0,1 inputs) inhibits the enzyme reaction, while keeping the optimum pH (assuming 0,0 and 1,1 input combinations) preserves a high enzyme activity. The challenging part of the present approach is the selection of an enzyme with a well-demonstrated bell-shape activity dependence on the pH value. While many enzymes can satisfy this condition, we selected pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase as this enzyme has the optimum pH center-located on the pH scale allowing the enzyme activity change by the acidic and basic pH shift from the optimum value corresponding to the highest activity. The present NXOR gate is added to the biomolecular “toolbox” as a new example of Boolean logic gates based on enzyme reactions.
Hydrogen peroxide (H2O2) is a typical surface sterilization agent for packaging materials used in the pharmaceutical, food and beverage industries. We use the finite-elements method to analyze the conceptual design of an in-line thermal evaporation unit to produce a heated gas mixture of air and evaporated H2O2 solution. For the numerical model, the required phase-transition variables of pure H2O2 solution and of the aerosol mixture are acquired from vapor-liquid equilibrium (VLE) diagrams derived from vapor-pressure formulations. This work combines homogeneous single-phase turbulent flow with heat-transfer physics to describe the operation of the evaporation unit. We introduce the apparent heat-capacity concept to approximate the non-isothermal phase-transition process of the H2O2-containing aerosol. Empirical and analytical functions are defined to represent the temperature- and pressure-dependent material properties of the aqueous H2O2 solution, the aerosol and the gas mixture. To validate the numerical model, the simulation results are compared to experimental data on the heating power required to produce the gas mixture. This shows good agreement with the deviations below 10%. Experimental observations on the formation of deposits due to the evaporation of stabilized H2O2 solution fits the prediction made from simulation results.
For performing point-of-care molecular diagnostics, magnetic immunoassays constitute a promising alternative to established enzyme-linked immunosorbent assays (ELISA) because they are fast, robust and sensitive. Simultaneous detection of multiple biomolecular targets from one body fluid sample is desired. The aim of this work is to show that multiplex magnetic immunodetection based on magnetic frequency mixing by means of modular immunofiltration columns prepared for different targets is feasible. By calculations of the magnetic response signal, the required spacing between the modules was determined. Immunofiltration columns were manufactured by 3D printing and antibody immobilization was performed in a batch approach. It was shown experimentally that two different target molecules in a sample solution could be individually detected in a single assaying step with magnetic measurements of the corresponding immobilization filters. The arrangement order of the filters and of a negative control did not influence the results. Thus, a simple and reliable approach to multi-target magnetic immunodetection was demonstrated.
A light-addressable potentiometric sensor (LAPS) is a field-effect-based (bio-) chemical sensor, in which a desired sensing area on the sensor surface can be defined by illumination. Light addressability can be used to visualize the concentration and spatial distribution of the target molecules, e.g., H+ ions. This unique feature has great potential for the label-free imaging of the metabolic activity of living organisms. The cultivation of those organisms needs specially tailored surface properties of the sensor. O2 plasma treatment is an attractive and promising tool for rapid surface engineering. However, the potential impacts of the technique are carefully investigated for the sensors that suffer from plasma-induced damage. Herein, a LAPS with a Ta2O5 pH-sensitive surface is successfully patterned by plasma treatment, and its effects are investigated by contact angle and scanning LAPS measurements. The plasma duration of 30 s (30 W) is found to be the threshold value, where excessive wettability begins. Furthermore, this treatment approach causes moderate plasma-induced damage, which can be reduced by thermal annealing (10 min at 300 °C). These findings provide a useful guideline to support future studies, where the LAPS surface is desired to be more hydrophilic by O2 plasma treatment.
Sensitive and rapid detection of cholera toxin subunit B using magnetic frequency mixing detection
(2019)
Cholera is a life-threatening disease caused by the cholera toxin (CT) as produced by some Vibrio cholerae serogroups. In this research we present a method which directly detects the toxin’s B subunit (CTB) in drinking water. For this purpose we performed a magnetic sandwich immunoassay inside a 3D immunofiltration column. We used two different commercially available antibodies to capture CTB and for binding to superparamagnetic beads. ELISA experiments were performed to select the antibody combination. The beads act as labels for the magnetic frequency mixing detection technique. We show that the limit of detection depends on the type of magnetic beads. A nonlinear Hill curve was fitted to the calibration measurements by means of a custom-written python software. We achieved a sensitive and rapid detection of CTB within a broad concentration range from 0.2 ng/ml to more
than 700 ng/ml.
The movement of magnetic beads due to a magnetic field gradient is of great interest in different application fields. In this report we present a technique based on a magnetic tweezers setup to measure the velocity factor of magnetically actuated individual superparamagnetic beads in a fluidic environment. Several beads can be tracked simultaneously in order to gain and improve statistics. Furthermore we show our results for different beads with hydrodynamic diameters between 200 and 1000 nm from diverse manufacturers. These measurement data can, for example, be used to determine design parameters for a magnetic separation system, like maximum flow rate and minimum separation time, or to select suitable beads for fixed experimental requirements.
In modern bioanalytical methods, it is often desired to detect several targets in one sample within one measurement. Immunological methods including those that use superparamagnetic beads are an important group of techniques for these applications. The goal of this work is to investigate the feasibility of simultaneously detecting different superparamagnetic beads acting as markers using the magnetic frequency mixing technique. The frequency of the magnetic excitation field is scanned while the lower driving frequency is kept constant. Due to the particles’ nonlinear magnetization, mixing frequencies are generated. To record their amplitude and phase information, a direct digitization of the pickup-coil’s signal with subsequent Fast Fourier Transformation is performed. By synchronizing both magnetic beads using frequency scanning in magnetic frequency mixing technique magnetic fields, a stable phase information is gained. In this research, it is shown that the amplitude of the dominant mixing component is proportional to the amount of superparamagnetic beads inside a sample. Additionally, it is shown that the phase does not show this behaviour. Excitation frequency scans of different bead types were performed, showing different phases, without correlation to their diverse amplitudes. Two commercially available beads were selected and a determination of their amount in a mixture is performed as a demonstration for multiplex measurements.
The enantioselective synthesis of α-hydroxy ketones and vicinal diols is an intriguing field because of the broad applicability of these molecules. Although, butandiol dehydrogenases are known to play a key role in the production of 2,3-butandiol, their potential as biocatalysts is still not well studied. Here, we investigate the biocatalytic properties of the meso-butanediol dehydrogenase from Bacillus licheniformis DSM 13T (BlBDH). The encoding gene was cloned with an N-terminal StrepII-tag and recombinantly overexpressed in E. coli. BlBDH is highly active towards several non-physiological diketones and α-hydroxyketones with varying aliphatic chain lengths or even containing phenyl moieties. By adjusting the reaction parameters in biotransformations the formation of either the α-hydroxyketone intermediate or the diol can be controlled.
α-hydroxy ketones (HK) and 1,2-diols are important building blocks for fine chemical synthesis. Here, we describe the R-selective 2,3-butanediol dehydrogenase from B. clausii DSM 8716ᵀ (BcBDH) that belongs to the metal-dependent medium chain dehydrogenases/reductases family (MDR) and catalyzes the selective asymmetric reduction of prochiral 1,2-diketones to the corresponding HK and, in some cases, the reduction of the same to the corresponding 1,2-diols. Aliphatic diketones, like 2,3-pentanedione, 2,3-hexanedione, 5-methyl-2,3-hexanedione, 3,4-hexanedione and 2,3-heptanedione are well transformed. In addition, surprisingly alkyl phenyl dicarbonyls, like 2-hydroxy-1-phenylpropan-1-one and phenylglyoxal are accepted, whereas their derivatives with two phenyl groups are not substrates. Supplementation of Mn²⁺ (1 mM) increases BcBDH's activity in biotransformations. Furthermore, the biocatalytic reduction of 5-methyl-2,3-hexanedione to mainly 5-methyl-3-hydroxy-2-hexanone with only small amounts of 5-methyl-2-hydroxy-3-hexanone within an enzyme membrane reactor is demonstrated.
LAPS-based monitoring of metabolic responses of bacterial cultures in a paper fermentation broth
(2020)
As an alternative renewable energy source, methane production in biogas plants is gaining more and more attention. Biomass in a bioreactor contains different types of microorganisms, which should be considered in terms of process-stability control. Metabolically inactive microorganisms within the fermentation process can lead to undesirable, time-consuming and cost-intensive interventions. Hence, monitoring of the cellular metabolism of bacterial populations in a fermentation broth is crucial to improve the biogas production, operation efficiency, and sustainability. In this work, the extracellular acidification of bacteria in a paper-fermentation broth is monitored after glucose uptake, utilizing a differential light-addressable potentiometric sensor (LAPS) system. The LAPS system is loaded with three different model microorganisms (Escherichia coli, Corynebacterium glutamicum, and Lactobacillus brevis) and the effect of the fermentation broth at different process stages on the metabolism of these bacteria is studied. In this way, different signal patterns related to the metabolic response of microorganisms can be identified. By means of calibration curves after glucose uptake, the overall extracellular acidification of bacterial populations within the fermentation process can be evaluated.
In this review article, we are going to present an overview on possible applications of light-addressable electrodes (LAE) as actuator/manipulation devices besides classical electrode structures. For LAEs, the electrode material consists of a semiconductor. Illumination with a light source with the appropiate wavelength leads to the generation of electron-hole pairs which can be utilized for further photoelectrochemical reaction. Due to recent progress in light-projection technologies, highly dynamic and flexible illumination patterns can be generated, opening new possibilities for light-addressable electrodes. A short introduction on semiconductor–electrolyte interfaces with light stimulation is given together with electrode-design approaches. Towards applications, the stimulation of cells with different electrode materials and fabrication designs is explained, followed by analyte-manipulation strategies and spatially resolved photoelectrochemical deposition of different material types.
Within the present work a sterilization process by a heated gas mixture that contains hydrogen peroxide (H₂O₂) is validated by experiments and numerical modeling techniques. The operational parameters that affect the sterilization efficacy are described alongside the two modes of sterilization: gaseous and condensed H₂O₂. Measurements with a previously developed H₂O₂ gas sensor are carried out to validate the applied H₂O₂ gas concentration during sterilization. We performed microbiological tests at different H₂O₂ gas concentrations by applying an end-point method to carrier strips, which contain different inoculation loads of Geobacillus stearothermophilus spores. The analysis of the sterilization process of a pharmaceutical glass vial is performed by numerical modeling. The numerical model combines heat- and advection-diffusion mass transfer with vapor–pressure equations to predict the location of condensate formation and the concentration of H₂O₂ at the packaging surfaces by changing the gas temperature. For a sterilization process of 0.7 s, a H₂O₂ gas concentration above 4% v/v is required to reach a log-count reduction above six. The numerical results showed the location of H₂O₂ condensate formation, which decreases with increasing sterilant-gas temperature. The model can be transferred to different gas nozzle- and packaging geometries to assure the absence of H₂O₂ residues.
Transport through Redox-Active Ru-Terpyridine Complexes Integrated in Single Nanoparticle Devices
(2020)
Transition metal complexes are electrofunctional molecules due to their high conductivity and their intrinsic switching ability involving a metal-to-ligand charge transfer. Here, a method is presented to contact reliably a few to single redox-active Ru-terpyridine complexes in a CMOS compatible nanodevice and preserve their electrical functionality. Using hybrid materials from 14 nm gold nanoparticles (AuNP) and bis-{4′-[4-(mercaptophenyl)-2,2′:6′,2″-terpyridine]}-ruthenium(II) complexes a device size of 30² nm² inclusive nanoelectrodes is achieved. Moreover, this method bears the opportunity for further downscaling. The Ru-complex AuNP devices show symmetric and asymmetric current versus voltage curves with a hysteretic characteristic in two well separated conductance ranges. By theoretical approximations based on the single-channel Landauer model, the charge transport through the formed double-barrier tunnel junction is thoroughly analyzed and its sensibility to the molecule/metal contact is revealed. It can be verified that tunneling transport through the HOMO is the main transport mechanism while decoherent hopping transport is present to a minor extent.
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.
Coronavirus disease 2019 (COVID-19) is a novel human infectious disease provoked by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, no specific vaccines or drugs against COVID-19 are available. Therefore, early diagnosis and treatment are essential in order to slow the virus spread and to contain the disease outbreak. Hence, new diagnostic tests and devices for virus detection in clinical samples that are faster, more accurate and reliable, easier and cost-efficient than existing ones are needed. Due to the small sizes, fast response time, label-free operation without the need for expensive and time-consuming labeling steps, the possibility of real-time and multiplexed measurements, robustness and portability (point-of-care and on-site testing), biosensors based on semiconductor field-effect devices (FEDs) are one of the most attractive platforms for an electrical detection of charged biomolecules and bioparticles by their intrinsic charge. In this review, recent advances and key developments in the field of label-free detection of viruses (including plant viruses) with various types of FEDs are presented. In recent years, however, certain plant viruses have also attracted additional interest for biosensor layouts: Their repetitive protein subunits arranged at nanometric spacing can be employed for coupling functional molecules. If used as adapters on sensor chip surfaces, they allow an efficient immobilization of analyte-specific recognition and detector elements such as antibodies and enzymes at highest surface densities. The display on plant viral bionanoparticles may also lead to long-time stabilization of sensor molecules upon repeated uses and has the potential to increase sensor performance substantially, compared to conventional layouts. This has been demonstrated in different proof-of-concept biosensor devices. Therefore, richly available plant viral particles, non-pathogenic for animals or humans, might gain novel importance if applied in receptor layers of FEDs. These perspectives are explained and discussed with regard to future detection strategies for COVID-19 and related viral diseases.
Extracellular acidification is a basic indicator for alterations in two vital metabolic pathways: glycolysis and cellular respiration. Measuring these alterations by monitoring extracellular acidification using cell-based biosensors such as LAPS plays an important role in studying these pathways whose disorders are associated with numerous diseases including cancer. However, the surface of the biosensors must be specially tailored to ensure high cell compatibility so that cells can represent more in vivo-like behavior, which is critical to gain more realistic in vitro results from the analyses, e.g., drug discovery experiments. In this work, O2 plasma patterning on the LAPS surface is studied to enhance surface features of the sensor chip, e.g., wettability and biofunctionality. The surface treated with O2 plasma for 30 s exhibits enhanced cytocompatibility for adherent CHO–K1 cells, which promotes cell spreading and proliferation. The plasma-modified LAPS chip is then integrated into a microfluidic system, which provides two identical channels to facilitate differential measurements of the extracellular acidification of CHO–K1 cells. To the best of our knowledge, it is the first time that extracellular acidification within microfluidic channels is quantitatively visualized as differential (bio-)chemical images.
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.
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.
Photoelectrochemical (PEC) biosensors are a rather novel type of biosensors thatutilizelighttoprovideinformationaboutthecompositionofananalyte,enablinglight-controlled multi-analyte measurements. For enzymatic PEC biosensors,amperometric detection principles are already known in the literature. In con-trast, there is only a little information on H+-ion sensitive PEC biosensors. Inthis work, we demonstrate the detection of H+ions emerged by H+-generatingenzymes, exemplarily demonstrated with penicillinase as a model enzyme on atitanium dioxide photoanode. First, we describe the pH sensitivity of the sensorand study possible photoelectrocatalytic reactions with penicillin. Second, weshow the enzymatic PEC detection of penicillin.
The treatment method to deactivate viable microorganisms from objects or products is termed sterilization. There are multiple forms of sterilization, each intended to be applied for a specific target, which depends on—but not limited to—the thermal, physical, and chemical stability of that target. Herein, an overview on the currently used sterilization processes in the global market is provided. Different sterilization techniques are grouped under a category that describes the method of treatment: radiation (gamma, electron beam, X-ray, and ultraviolet), thermal (dry and moist heat), and chemical (ethylene oxide, ozone, chlorine dioxide, and hydrogen peroxide). For each sterilization process, the typical process parameters as defined by regulations and the mode of antimicrobial activity are summarized. Finally, the recommended microorganisms that are used as biological indicators to validate sterilization processes in accordance with the rules that are established by various regulatory agencies are summarized.