@article{JablonskiPoghossianKeusgenetal.2021, author = {Jablonski, Melanie and Poghossian, Arshak and Keusgen, Michael and Wege, Christina and Sch{\"o}ning, Michael Josef}, title = {Detection of plant virus particles with a capacitive field-effect sensor}, series = {Analytical and Bioanalytical Chemistry}, volume = {413}, journal = {Analytical and Bioanalytical Chemistry}, publisher = {Springer Nature}, address = {Cham}, issn = {1618-2650}, doi = {10.1007/s00216-021-03448-8}, pages = {5669 -- 5678}, year = {2021}, abstract = {Plant viruses are major contributors to crop losses and induce high economic costs worldwide. For reliable, on-site and early detection of plant viral diseases, portable biosensors are of great interest. In this study, a field-effect SiO2-gate electrolyte-insulator-semiconductor (EIS) sensor was utilized for the label-free electrostatic detection of tobacco mosaic virus (TMV) particles as a model plant pathogen. The capacitive EIS sensor has been characterized regarding its TMV sensitivity by means of constant-capacitance method. The EIS sensor was able to detect biotinylated TMV particles from a solution with a TMV concentration as low as 0.025 nM. A good correlation between the registered EIS sensor signal and the density of adsorbed TMV particles assessed from scanning electron microscopy images of the SiO2-gate chip surface was observed. Additionally, the isoelectric point of the biotinylated TMV particles was determined via zeta potential measurements and the influence of ionic strength of the measurement solution on the TMV-modified EIS sensor signal has been studied.}, language = {en} } @article{PoghossianKarschuckWagneretal.2022, author = {Poghossian, Arshak and Karschuck, Tobias and Wagner, Patrick and Sch{\"o}ning, Michael Josef}, title = {Field-Effect Capacitors Decorated with Ligand-Stabilized Gold Nanoparticles: Modeling and Experiments}, series = {Biosensors}, volume = {12}, journal = {Biosensors}, number = {5}, publisher = {MDPI}, address = {Basel}, issn = {2079-6374}, doi = {10.3390/bios12050334}, pages = {Artikel 334}, year = {2022}, abstract = {Nanoparticles are recognized as highly attractive tunable materials for designing field-effect biosensors with enhanced performance. In this work, we present a theoretical model for electrolyte-insulator-semiconductor capacitors (EISCAP) decorated with ligand-stabilized charged gold nanoparticles. The charged AuNPs are taken into account as additional, nanometer-sized local gates. The capacitance-voltage (C-V) curves and constant-capacitance (ConCap) signals of the AuNP-decorated EISCAPs have been simulated. The impact of the AuNP coverage on the shift of the C-V curves and the ConCap signals was also studied experimentally on Al-p-Si-SiO₂ EISCAPs decorated with positively charged aminooctanethiol-capped AuNPs. In addition, the surface of the EISCAPs, modified with AuNPs, was characterized by scanning electron microscopy for different immobilization times of the nanoparticles.}, language = {en} } @article{PourshahidiAchtsnichtOffenhaeusseretal.2022, author = {Pourshahidi, Ali Mohammad and Achtsnicht, Stefan and Offenh{\"a}usser, Andreas and Krause, Hans-Joachim}, title = {Frequency Mixing Magnetic Detection Setup Employing Permanent Ring Magnets as a Static Offset Field Source}, series = {Sensors}, volume = {22}, journal = {Sensors}, number = {22}, editor = {Offenh{\"a}usser, Andreas}, publisher = {MDPI}, address = {Basel}, issn = {1424-8220}, doi = {10.3390/s22228776}, pages = {12 Seiten}, year = {2022}, abstract = {Frequency mixing magnetic detection (FMMD) has been explored for its applications in fields of magnetic biosensing, multiplex detection of magnetic nanoparticles (MNP) and the determination of core size distribution of MNP samples. Such applications rely on the application of a static offset magnetic field, which is generated traditionally with an electromagnet. Such a setup requires a current source, as well as passive or active cooling strategies, which directly sets a limitation based on the portability aspect that is desired for point of care (POC) monitoring applications. In this work, a measurement head is introduced that involves the utilization of two ring-shaped permanent magnets to generate a static offset magnetic field. A steel cylinder in the ring bores homogenizes the field. By variation of the distance between the ring magnets and of the thickness of the steel cylinder, the magnitude of the magnetic field at the sample position can be adjusted. Furthermore, the measurement setup is compared to the electromagnet offset module based on measured signals and temperature behavior.}, language = {en} } @article{KarschuckKaulenPoghossianetal.2021, author = {Karschuck, Tobias and Kaulen, Corinna and Poghossian, Arshak and Wagner, Patrick H. and Sch{\"o}ning, Michael Josef}, title = {Gold nanoparticle-modified capacitive field-effect sensors: Studying the surface density of nanoparticles and coupling of charged polyelectrolyte macromolecules}, series = {Electrochemical Science Advances}, volume = {2}, journal = {Electrochemical Science Advances}, number = {5}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {0938-5193}, doi = {10.1002/elsa.202100179}, pages = {10 Seiten}, year = {2021}, abstract = {The coupling of ligand-stabilized gold nanoparticles with field-effect devices offers new possibilities for label-free biosensing. In this work, we study the immobilization of aminooctanethiol-stabilized gold nanoparticles (AuAOTs) on the silicon dioxide surface of a capacitive field-effect sensor. The terminal amino group of the AuAOT is well suited for the functionalization with biomolecules. The attachment of the positively-charged AuAOTs on a capacitive field-effect sensor was detected by direct electrical readout using capacitance-voltage and constant capacitance measurements. With a higher particle density on the sensor surface, the measured signal change was correspondingly more pronounced. The results demonstrate the ability of capacitive field-effect sensors for the non-destructive quantitative validation of nanoparticle immobilization. In addition, the electrostatic binding of the polyanion polystyrene sulfonate to the AuAOT-modified sensor surface was studied as a model system for the label-free detection of charged macromolecules. Most likely, this approach can be transferred to the label-free detection of other charged molecules such as enzymes or antibodies.}, language = {en} } @article{VahidpourGuthmanArreolaetal.2022, author = {Vahidpour, Farnoosh and Guthman, Eric and Arreola, Julia and Alghazali, Yousef H. M. and Wagner, Torsten and Sch{\"o}ning, Michael Josef}, title = {Assessment of Various Process Parameters for Optimized Sterilization Conditions Using a Multi-Sensing Platform}, series = {Foods}, volume = {11}, journal = {Foods}, number = {5}, publisher = {MDPI}, address = {Basel}, issn = {2304-8158}, doi = {10.3390/foods11050660}, pages = {Artikel 660}, year = {2022}, abstract = {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.}, language = {en} } @article{PoghossianWeldenBuniatyanetal.2021, author = {Poghossian, Arshak and Welden, Rene and Buniatyan, Vahe V. and Sch{\"o}ning, Michael Josef}, title = {An Array of On-Chip Integrated, Individually Addressable Capacitive Field-Effect Sensors with Control Gate: Design and Modelling}, series = {Sensors}, volume = {21}, journal = {Sensors}, number = {18}, publisher = {MDPI}, address = {Basel}, issn = {1424-8220}, doi = {10.3390/s21186161}, pages = {17}, year = {2021}, abstract = {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.}, language = {en} } @article{PourshahidiAchtsnichtNambipareecheeetal.2021, author = {Pourshahidi, Ali Mohammad and Achtsnicht, Stefan and Nambipareechee, Mrinal Murali and Offenh{\"a}usser, Andreas and Krause, Hans-Joachim}, title = {Multiplex detection of magnetic beads using offset field dependent frequency mixing magnetic detection}, series = {Sensors}, volume = {21}, journal = {Sensors}, number = {17}, publisher = {MDPI}, address = {Basel}, issn = {1424-8220}, doi = {10.3390/s21175859}, pages = {16 Seiten}, year = {2021}, abstract = {Magnetic immunoassays employing Frequency Mixing Magnetic Detection (FMMD) have recently become increasingly popular for quantitative detection of various analytes. Simultaneous analysis of a sample for two or more targets is desirable in order to reduce the sample amount, save consumables, and save time. We show that different types of magnetic beads can be distinguished according to their frequency mixing response to a two-frequency magnetic excitation at different static magnetic offset fields. We recorded the offset field dependent FMMD response of two different particle types at frequencies ƒ₁ + n⋅ƒ₂, n = 1, 2, 3, 4 with ƒ₁ = 30.8 kHz and ƒ₂ = 63 Hz. Their signals were clearly distinguishable by the locations of the extremes and zeros of their responses. Binary mixtures of the two particle types were prepared with different mixing ratios. The mixture samples were analyzed by determining the best linear combination of the two pure constituents that best resembled the measured signals of the mixtures. Using a quadratic programming algorithm, the mixing ratios could be determined with an accuracy of greater than 14\%. If each particle type is functionalized with a different antibody, multiplex detection of two different analytes becomes feasible.}, language = {en} } @article{HaegerGrankinWagner2023, author = {Haeger, Gerrit and Grankin, Alina and Wagner, Michaela}, title = {Construction of an Aspergillus oryzae triple amylase deletion mutant as a chassis to evaluate industrially relevant amylases using multiplex CRISPR/Cas9 editing technology}, series = {Applied Research}, journal = {Applied Research}, number = {Early View}, publisher = {Wiley-VCH}, issn = {2702-4288}, doi = {10.1002/appl.202200106}, pages = {1 -- 15}, year = {2023}, abstract = {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.}, language = {en} } @article{VahidpourAlghazaliAkcaetal.2022, author = {Vahidpour, Farnoosh and Alghazali, Yousef and Akca, Sevilay and Hommes, Gregor and Sch{\"o}ning, Michael Josef}, title = {An Enzyme-Based Interdigitated Electrode-Type Biosensor for Detecting Low Concentrations of H₂O₂ Vapor/Aerosol}, series = {Chemosensors}, volume = {10}, journal = {Chemosensors}, number = {6}, publisher = {MDPI}, address = {Basel}, issn = {2227-9040}, doi = {10.3390/chemosensors10060202}, pages = {Arikel 202}, year = {2022}, abstract = {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.}, language = {en} } @article{HaegerJolmesOyenetal.2024, author = {Haeger, Gerrit and Jolmes, Tristan and Oyen, Sven and Jaeger, Karl-Erich and Bongaerts, Johannes and Sch{\"o}rken, Ulrich and Siegert, Petra}, title = {Novel recombinant aminoacylase from Paraburkholderia monticola capable of N-acyl-amino acid synthesis}, series = {Applied Microbiology and Biotechnology}, journal = {Applied Microbiology and Biotechnology}, number = {108}, publisher = {Springer}, address = {Berlin}, issn = {1432-0614}, doi = {10.1007/s00253-023-12868-8}, pages = {14 Seiten}, year = {2024}, abstract = {N-Acyl-amino acids can act as mild biobased surfactants, which are used, e.g., in baby shampoos. However, their chemical synthesis needs acyl chlorides and does not meet sustainability criteria. Thus, the identification of biocatalysts to develop greener synthesis routes is desirable. We describe a novel aminoacylase from Paraburkholderia monticola DSM 100849 (PmAcy) which was identified, cloned, and evaluated for its N-acyl-amino acid synthesis potential. Soluble protein was obtained by expression in lactose autoinduction medium and co-expression of molecular chaperones GroEL/S. Strep-tag affinity purification enriched the enzyme 16-fold and yielded 15 mg pure enzyme from 100 mL of culture. Biochemical characterization revealed that PmAcy possesses beneficial traits for industrial application like high temperature and pH-stability. A heat activation of PmAcy was observed upon incubation at temperatures up to 80 °C. Hydrolytic activity of PmAcy was detected with several N-acyl-amino acids as substrates and exhibited the highest conversion rate of 773 U/mg with N-lauroyl-L-alanine at 75 °C. The enzyme preferred long-chain acyl-amino-acids and displayed hardly any activity with acetyl-amino acids. PmAcy was also capable of N-acyl-amino acid synthesis with good conversion rates. The best synthesis results were obtained with the cationic L-amino acids L-arginine and L-lysine as well as with L-leucine and L-phenylalanine. Exemplarily, L-phenylalanine was acylated with fatty acids of chain lengths from C8 to C18 with conversion rates of up to 75\%. N-lauroyl-L-phenylalanine was purified by precipitation, and the structure of the reaction product was verified by LC-MS and NMR.}, language = {en} } @article{HaegerWirgesTanzmannetal.2023, author = {Haeger, Gerrit and Wirges, Jessika and Tanzmann, Nicole and Oyen, Sven and Jolmes, Tristan and Jaeger, Karl-Erich and Sch{\"o}rken, Ulrich and Bongaerts, Johannes and Siegert, Petra}, title = {Chaperone assisted recombinant expression of a mycobacterial aminoacylase in Vibrio natriegens and Escherichia coli capable of N-lauroyl-L-amino acid synthesis}, series = {Microbial Cell Factories}, journal = {Microbial Cell Factories}, number = {22}, publisher = {Springer Nature}, issn = {1475-2859}, doi = {10.1186/s12934-023-02079-1}, pages = {Article number: 77 (2023)}, year = {2023}, abstract = {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.}, language = {en} } @techreport{HaegerBongaertsSiegert2023, author = {Haeger, Gerrit and Bongaerts, Johannes and Siegert, Petra}, title = {Abschlussbericht Teil II: Eingehende Darstellung Neue biobasierte Lipopeptide aus nachhaltiger Produktion (LipoPep)}, pages = {17Seiten}, year = {2023}, language = {de} } @techreport{SiegertBongaertsWagneretal.2022, author = {Siegert, Petra and Bongaerts, Johannes and Wagner, Torsten and Sch{\"o}ning, Michael Josef and Selmer, Thorsten}, title = {Abschlussbericht zum Projekt zur {\"U}berwachung biotechnologischer Prozesse mittels Diacetyl-/Acetoin-Biosensor und Evaluierung von Acetoin-Reduktasen zur Verwendung in Biotransformationen}, address = {Aachen}, organization = {FH Aachen}, pages = {16 Seiten}, year = {2022}, language = {de} } @article{JanusAchtsnichtTempeletal.2023, author = {Janus, Kevin Alexander and Achtsnicht, Stefan and Tempel, Laura and Drinic, Aleksaner and Kopp, Alexander and Keusgen, Michael and Sch{\"o}ning, Michael Josef}, title = {Influence of fibroin membrane composition and curing parameters on the performance of a biodegradable enzymatic biosensor manufactured from Silicon-Free Carbon}, series = {Physica status solidi : pss. A, Applications and materials science}, volume = {220}, journal = {Physica status solidi : pss. A, Applications and materials science}, number = {22}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1862-6300 (Print)}, doi = {10.1002/pssa.202300081}, pages = {Artikel 2300081}, year = {2023}, abstract = {Herein, fibroin, polylactide (PLA), and carbon are investigated for their suitability as biocompatible and biodegradable materials for amperometric biosensors. For this purpose, screen-printed carbon electrodes on the biodegradable substrates fibroin and PLA are modified with a glucose oxidase membrane and then encapsulated with the biocompatible material Ecoflex. The influence of different curing parameters of the carbon electrodes on the resulting biosensor characteristics is studied. The morphology of the electrodes is investigated by scanning electron microscopy, and the biosensor performance is examined by amperometric measurements of glucose (0.5-10 mM) in phosphate buffer solution, pH 7.4, at an applied potential of 1.2 V versus a Ag/AgCl reference electrode. Instead of Ecoflex, fibroin, PLA, and wound adhesive are tested as alternative encapsulation compounds: a series of swelling tests with different fibroin compositions, PLA, and Ecoflex has been performed before characterizing the most promising candidates by chronoamperometry. Therefore, the carbon electrodes are completely covered with the particular encapsulation material. Chronoamperometric measurements with H2O2 concentrations between 0.5 and 10 mM enable studying the leakage current behavior.}, language = {en} } @phdthesis{Achtsnicht2020, author = {Achtsnicht, Stefan}, title = {Multiplex-Magnetdetektion von superparamagnetischen Beads zur Identifizierung von Trinkwasserkontaminationen}, doi = {10.18154/RWTH-2020-12052}, pages = {144 Seiten}, year = {2020}, abstract = {Die qualitative und quantitative Detektion von Zielsubstanzen innerhalb einer w{\"a}ssrigen Probe ist f{\"u}r viele Fragestellungen von Interesse, etwa bei der Detektion von Kontaminationen in Trinkwasser in Krisensituationen. Hierbei ist es nicht nur wichtig, dass Pathogene m{\"o}glichst sensitiv detektiert werden k{\"o}nnen, sondern auch, dass die Analyse schnell erfolgt, um Betroffenen im Katastrophenfall z{\"u}gig sicheres Trinkwasser zu Verf{\"u}gung stellen zu k{\"o}nnen. Da bei einem solchen Szenario nicht von einer in der N{\"a}he befindlichen funktionierenden Laborinfrastruktur ausgegangen werden kann, ist es wichtig, dass die Messung direkt vor Ort erfolgen kann. Im Rahmen dieser Arbeit wurde untersucht, ob eine derartige Schnellanalytik mithilfe von superparamagnetischen Beads (MBs) und der magnetischen Frequenzmischtechnik m{\"o}glich ist. Dabei werden die MBs mit Hilfe von prim{\"a}ren Antik{\"o}rpern an die Zielsubstanz gebunden und mit sekund{\"a}ren Antik{\"o}rpern an die Poren-Oberfl{\"a}che eines Polyethylen-Filters fixiert (Sandwich-Immunoassay). So kann die Quantifizierung der Zielsubstanz auf eine magnetische Messung der immobilisierten MB-Marker zur{\"u}ckgef{\"u}hrt werden. Die magnetische Frequenzmischtechnik basiert auf der Anregung der Probe mit Magnetfeldern zweier verschiedener Frequenzen. Die durch die nichtlineare Magnetisierungsform der superparamagnetischen MBs entstehenden Mischfrequenzen werden typischerweise mithilfe einer zweistufigen Lock-in-Detektion analysiert (analoge Demodulation), die in einem Magnetreader als Handheldger{\"a}t realisiert wurde. Zus{\"a}tzlich zu dieser Technik wurde das Prinzip der direkten Digitalisierung des gesamten Antwortsignals mit anschließender Fourier-Analyse der erzeugten Mischfrequenzen experimentell umgesetzt, um die Amplituden und Phasen mehrerer Mischfrequenzen simultan zu erfassen. Eine M{\"o}glichkeit zur Sensitivit{\"a}tssteigerung ist die magnetische Aufkonzentration, indem vor der magnetischen Analyse eine Separation der MBs aus einem gr{\"o}ßeren Probenvolumen mittels magnetischem Feldgradienten durchgef{\"u}hrt wird. Zur Charakterisierung verschiedener kommerzieller MBs hinsichtlich ihrer magnetischen Separierbarkeit wurde ein Aufbau zur Messung ihrer magnetophoretischen Beweglichkeiten realisiert und ihre Geschwindigkeiten im Gradientenfeld mikroskopisch gemessen.Da eine Probe oftmals nicht nur auf eine einzige Zielsubstanz, sondern simultan auf mehrere verschiedene Pathogene hin untersucht werden soll, wurden verschiedene Ans{\"a}tze entwickelt und getestet, die einen solchen multiparametrischen magnetischen Immunoassay erm{\"o}glichen. Einerseits wurde eine r{\"a}umliche Separation der Bindungsbereiche f{\"u}r verschiedene Zielsubstanzen realisiert, die sequentiell ausgewertet werden k{\"o}nnen. Andererseits wurde die Unterscheidung von verschiedenen Zielsubstanzen anhand der Charakteristika der an sie gebundenen, verschieden funktionalisierten MB-Typen untersucht. F{\"u}r eine solche Unterscheidung wurde zum einen die Anregefrequenz der magnetischen Frequenzmischtechnik w{\"a}hrend einer Messung variiert. Damit konnte gezeigt werden, dass sich verschiedene MB-Sorten anhand der Phase ihrer Frequenzmischsignale voneinander unterscheiden lassen. Weiterhin wurde gezeigt, dass sich der Signalverlauf einer bin{\"a}ren Mischung zweier verschiedener MB-Typen als gradueller {\"U}bergang der Verl{\"a}ufe der beiden reinen MB-L{\"o}sungen ergibt. Eine weitere Analysemethode f{\"u}r einen multiparametrischen Immunoassay besteht darin, ein zus{\"a}tzliches einstellbares statisches magnetisches Offsetfeld zu verwenden. Hierf{\"u}r wurden mehrere Aufbauten auf Basis von Permanent- und Elektromagneten simuliert, konstruiert und charakterisiert. Mithilfe von Simulationen konnte gezeigt werden, dass eine auf diesem Verfahren beruhende Unterscheidung f{\"u}r MBs mit unterschiedlichen magnetischen Partikelmomenten m{\"o}glich ist. Als direkte Anwendung des hier entwickelten Magnetreaders in Zusammenspiel mit der digitalen Demodulation wurde ein magnetischer Assay gegen die B-Untereinheit des Choleratoxins in Trinkwasser mit einem niedrigen Detektionslimit von 0,2 ng/ml demonstriert.}, language = {de} } @article{AchtsnichtPourshahidiOffenhaeusseretal.2019, author = {Achtsnicht, Stefan and Pourshahidi, Ali Mohammad and Offenh{\"a}usser, Andreas and Krause, Hans-Joachim}, title = {Multiplex detection of different magnetic beads using frequency scanning in magnetic frequency mixing technique}, series = {Sensors}, volume = {19}, journal = {Sensors}, number = {11}, publisher = {MDPI}, address = {Basel}, issn = {1424-8220}, doi = {10.3390/s19112599}, pages = {13 Seiten}, year = {2019}, abstract = {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.}, language = {en} } @article{FalkenbergVossBottetal.2023, author = {Falkenberg, Fabian and Voß, Leonie and Bott, Michael and Bongaerts, Johannes and Siegert, Petra}, title = {New robust subtilisins from halotolerant and halophilic Bacillaceae}, series = {Applied Microbiology and Biotechnology}, volume = {107}, journal = {Applied Microbiology and Biotechnology}, publisher = {Springer Nature}, address = {Berlin}, issn = {1432-0614}, doi = {10.1007/s00253-023-12553-w}, pages = {3939 -- 3954}, year = {2023}, abstract = {The aim of the present study was the characterisation of three true subtilisins and one phylogenetically intermediate subtilisin from halotolerant and halophilic microorganisms. Considering the currently growing enzyme market for efficient and novel biocatalysts, data mining is a promising source for novel, as yet uncharacterised enzymes, especially from halophilic or halotolerant Bacillaceae, which offer great potential to meet industrial needs. Both halophilic bacteria Pontibacillus marinus DSM 16465ᵀ and Alkalibacillus haloalkaliphilus DSM 5271ᵀ and both halotolerant bacteria Metabacillus indicus DSM 16189 and Litchfieldia alkalitelluris DSM 16976ᵀ served as a source for the four new subtilisins SPPM, SPAH, SPMI and SPLA. The protease genes were cloned and expressed in Bacillus subtilis DB104. Purification to apparent homogeneity was achieved by ethanol precipitation, desalting and ion-exchange chromatography. Enzyme activity could be observed between pH 5.0-12.0 with an optimum for SPPM, SPMI and SPLA around pH 9.0 and for SPAH at pH 10.0. The optimal temperature for SPMI and SPLA was 70 °C and for SPPM and SPAH 55 °C and 50 °C, respectively. All proteases showed high stability towards 5\% (w/v) SDS and were active even at NaCl concentrations of 5 M. The four proteases demonstrate potential for future biotechnological applications.}, language = {en} } @article{AchtsnichtToedterNiehuesetal.2019, author = {Achtsnicht, Stefan and T{\"o}dter, Julia and Niehues, Julia and Tel{\"o}ken, Matthias and Offenh{\"a}usser, Andreas and Krause, Hans-Joachim and Schr{\"o}per, Florian}, title = {3D printed modular immunofiltration columns for frequency mixing-based multiplex magnetic immunodetection}, series = {Sensors}, volume = {19}, journal = {Sensors}, number = {1}, publisher = {MDPI}, address = {Basel}, issn = {1424-8220}, doi = {10.3390/s19010148}, pages = {15 Seiten}, year = {2019}, abstract = {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.}, language = {en} } @article{AchtsnichtSchoenenbornOffenhaeusseretal.2019, author = {Achtsnicht, Stefan and Sch{\"o}nenborn, Kristina and Offenh{\"a}usser, Andreas and Krause, Hans-Joachim}, title = {Measurement of the magnetophoretic velocity of different superparamagnetic beads}, series = {Journal of Magnetism and Magnetic Materials}, volume = {477}, journal = {Journal of Magnetism and Magnetic Materials}, number = {1}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0304-8853}, doi = {10.1016/j.jmmm.2018.10.066}, pages = {244 -- 248}, year = {2019}, abstract = {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.}, language = {en} } @article{RabehiGarlanAchtsnichtetal.2018, author = {Rabehi, Amine and Garlan, Benjamin and Achtsnicht, Stefan and Krause, Hans-Joachim and Offenh{\"a}usser, Andreas and Ngo, Kieu and Neveu, Sophie and Graff-Dubois, Stephanie and Kokabi, Hamid}, title = {Magnetic detection structure for Lab-on-Chip applications based on the frequency mixing technique}, series = {Sensors}, volume = {18}, journal = {Sensors}, number = {6}, publisher = {MDPI}, address = {Basel}, issn = {1424-8220}, doi = {10.3390/s18061747}, pages = {14 Seiten}, year = {2018}, abstract = {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.}, language = {en} }