TY - JOUR A1 - Muschallik, Lukas A1 - Molinnus, Denise A1 - Bongaerts, Johannes A1 - Pohl, Martina A1 - Wagner, Torsten A1 - Schöning, Michael Josef A1 - Siegert, Petra A1 - Selmer, Thorsten T1 - (R,R)-Butane-2,3-diol Dehydrogenase from Bacillus clausii DSM 8716T: Cloning and Expression of the bdhA-Gene, and Initial Characterization of Enzyme JF - Journal of Biotechnology N2 - The gene encoding a putative (R,R)-butane-2,3-diol dehydrogenase (bdhA) from Bacillus clausii DSM 8716T was isolated, sequenced and expressed in Escherichia coli. The amino acid sequence of the encoded protein is only distantly related to previously studied enzymes (identity 33–43%) and exhibited some uncharted peculiarities. An N-terminally StrepII-tagged enzyme variant was purified and initially characterized. The isolated enzyme catalyzed the (R)-specific oxidation of (R,R)- and meso-butane-2,3-diol to (R)- and (S)-acetoin with specific activities of 12 U/mg and 23 U/mg, respectively. Likewise, racemic acetoin was reduced with a specific activity of up to 115 U/mg yielding a mixture of (R,R)- and meso-butane-2,3-diol, while the enzyme reduced butane-2,3-dione (Vmax 74 U/mg) solely to (R,R)-butane-2,3-diol via (R)-acetoin. For these reactions only activity with the co-substrates NADH/NAD+ was observed. The enzyme accepted a selection of vicinal diketones, α-hydroxy ketones and vicinal diols as alternative substrates. Although the physiological function of the enzyme in B. clausii remains elusive, the data presented herein clearly demonstrates that the encoded enzyme is a genuine (R,R)-butane-2,3-diol dehydrogenase with potential for applications in biocatalysis and sensor development. Y1 - 2017 U6 - http://dx.doi.org/10.1016/j.jbiotec.2017.07.020 SN - 0168-1656 VL - 258 SP - 41 EP - 50 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Oliveira, Danilo A. A1 - Molinnus, Denise A1 - Beging, Stefan A1 - Siqueira Jr, José R. A1 - Schöning, Michael Josef T1 - Biosensor Based on Self-Assembled Films of Graphene Oxide and Polyaniline Using a Field-Effect Device Platform JF - physica status solidi (a) applications and materials science N2 - 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. KW - capacitive electrolyte–insulator–semiconductor sensors KW - graphene oxide KW - layer-by-layer technique KW - nanomaterials KW - polyaniline Y1 - 2021 U6 - http://dx.doi.org/10.1002/pssa.202000747 SN - 1862-6319 N1 - Corresponding author: José R. Siqueira Jr & Michael J. Schöning VL - 218 IS - 13 SP - 1 EP - 9 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Jablonski, Melanie A1 - Münstermann, Felix A1 - Nork, Jasmina A1 - Molinnus, Denise A1 - Muschallik, Lukas A1 - Bongaerts, Johannes A1 - Wagner, Torsten A1 - Keusgen, Michael A1 - Siegert, Petra A1 - Schöning, Michael Josef T1 - Capacitive field‐effect biosensor applied for the detection of acetoin in alcoholic beverages and fermentation broths JF - physica status solidi (a) applications and materials science N2 - 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. KW - acetoin KW - acetoin reductase KW - alcoholic beverages KW - biosensors KW - capacitive field-effect sensors Y1 - 2021 U6 - http://dx.doi.org/10.1002/pssa.202000765 SN - 1862-6319 VL - 218 IS - 13 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Molinnus, Denise A1 - Hardt, G. A1 - Käver, L. A1 - Willenberg, H.S. A1 - Kröger, J.-C. A1 - Poghossian, Arshak A1 - Keusgen, Michael A1 - Schöning, Michael Josef T1 - Chip-based biosensor for the detection of low adrenaline concentrations to support adrenal venous sampling JF - Sensor and Actuators B: Chemical N2 - 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. Y1 - 2018 U6 - http://dx.doi.org/10.1016/j.snb.2018.05.136 SN - 0925-4005 VL - 272 SP - 21 EP - 27 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Molinnus, Denise A1 - Bäcker, Matthias A1 - Iken, Heiko A1 - Poghossian, Arshak A1 - Keusgen, Michael A1 - Schöning, Michael Josef T1 - Concept for a biomolecular logic chip with an integrated sensor and actuator function JF - Physica status solidi (a) N2 - 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. Y1 - 2015 U6 - http://dx.doi.org/10.1002/pssa.201431913 SN - 1862-6319 VL - 212 IS - 6 SP - 1382 EP - 1388 PB - Wiley CY - Weinheim ER - TY - JOUR A1 - Molinnus, Denise A1 - Poghossian, Arshak A1 - Keusgen, Michael A1 - Katz, Evgeny A1 - Schöning, Michael Josef T1 - Coupling of Biomolecular Logic Gates with Electronic Transducers: From Single Enzyme Logic Gates to Sense/Act/Treat Chips JF - Electroanalysis N2 - The integration of biomolecular logic principles with electronic transducers allows designing novel digital biosensors with direct electrical output, logically triggered drug-release, and closed-loop sense/act/treat systems. This opens new opportunities for advanced personalized medicine in the context of theranostics. In the present work, we will discuss selected examples of recent developments in the field of interfacing enzyme logic gates with electrodes and semiconductor field-effect devices. Special attention is given to an enzyme OR/Reset logic gate based on a capacitive field-effect electrolyte-insulator-semiconductor sensor modified with a multi-enzyme membrane. Further examples are a digital adrenaline biosensor based on an AND logic gate with binary YES/NO output and an integrated closed-loop sense/act/treat system comprising an amperometric glucose sensor, a hydrogel actuator, and an insulin (drug) sensor. Y1 - 2017 U6 - http://dx.doi.org/10.1002/elan.201700208 SN - 1521-4109 VL - 29 IS - 8 SP - 1840 EP - 1849 PB - Wiley CY - Weinheim ER - TY - CHAP A1 - Molinnus, Denise A1 - Hardt, Gabriel A1 - Käver, Larissa A1 - Willenberg, Holger S. A1 - Poghossian, Arshak A1 - Keusgen, Michael A1 - Schöning, Michael Josef T1 - Detection of Adrenaline Based on Bioelectrocatalytical System to Support Tumor Diagnostic Technology T2 - MDPI Proceedings Y1 - 2017 U6 - http://dx.doi.org/10.3390/proceedings1040506 ER - TY - JOUR A1 - Molinnus, Denise A1 - Bäcker, Matthias A1 - Siegert, Petra A1 - Willenberg, H. A1 - Poghossian, Arshak A1 - Keusgen, M. A1 - Schöning, Michael Josef T1 - Detection of Adrenaline Based on Substrate Recycling Amplification JF - Procedia Engineering N2 - An amperometric enzyme biosensor has been applied for the detection of adrenaline. The adrenaline biosensor has been prepared by modification of an oxygen electrode with the enzyme laccase that operates at a broad pH range between pH 3.5 to pH 8. The enzyme molecules were immobilized via cross-linking with glutaraldehyde. The sensitivity of the developed adrenaline biosensor in different pH buffer solutions has been studied. Y1 - 2015 U6 - http://dx.doi.org/10.1016/j.proeng.2015.08.708 SN - 1877-7058 N1 - Eurosensors 2015 VL - 120 SP - 540 EP - 543 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Molinnus, Denise A1 - Hardt, Gabriel A1 - Siegert, Petra A1 - Willenberg, Holger S. A1 - Poghossian, Arshak A1 - Keusgen, Michael A1 - Schöning, Michael Josef T1 - Detection of Adrenaline in Blood Plasma as Biomarker for Adrenal Venous Sampling JF - Electroanalysis N2 - 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. Y1 - 2018 U6 - http://dx.doi.org/10.1002/elan.201800026 SN - 1521-4109 VL - 30 IS - 5 SP - 937 EP - 942 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Molinnus, Denise A1 - Muschallik, Lukas A1 - Gonzalez, Laura Osorio A1 - Bongaerts, Johannes A1 - Wagner, Torsten A1 - Selmer, Thorsten A1 - Siegert, Petra A1 - Keusgen, Michael A1 - Schöning, Michael Josef T1 - Development and characterization of a field-effect biosensor for the detection of acetoin JF - Biosensors and Bioelectronics N2 - 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. Y1 - 2018 U6 - http://dx.doi.org/10.1016/j.bios.2018.05.023 VL - 115 SP - 1 EP - 6 PB - Elsevier CY - Amsterdam ER -