TY - JOUR A1 - Karschuck, Tobias A1 - Poghossian, Arshak A1 - Ser, Joey A1 - Tsokolakyan, Astghik A1 - Achtsnicht, Stefan A1 - Wagner, Patrick A1 - Schöning, Michael Josef T1 - Capacitive model of enzyme-modified field-effect biosensors: Impact of enzyme coverage JF - Sensors and Actuators B: Chemical N2 - Electrolyte-insulator-semiconductor capacitors (EISCAP) belong to field-effect sensors having an attractive transducer architecture for constructing various biochemical sensors. In this study, a capacitive model of enzyme-modified EISCAPs has been developed and the impact of the surface coverage of immobilized enzymes on its capacitance-voltage and constant-capacitance characteristics was studied theoretically and experimentally. The used multicell arrangement enables a multiplexed electrochemical characterization of up to sixteen EISCAPs. Different enzyme coverages have been achieved by means of parallel electrical connection of bare and enzyme-covered single EISCAPs in diverse combinations. As predicted by the model, with increasing the enzyme coverage, both the shift of capacitance-voltage curves and the amplitude of the constant-capacitance signal increase, resulting in an enhancement of analyte sensitivity of the EISCAP biosensor. In addition, the capability of the multicell arrangement with multi-enzyme covered EISCAPs for sequentially detecting multianalytes (penicillin and urea) utilizing the enzymes penicillinase and urease has been experimentally demonstrated and discussed. KW - Field-effect biosensor KW - Capacitive model KW - Enzyme coverage KW - Multianalyte detection KW - Penicillin Y1 - 2024 U6 - http://dx.doi.org/10.1016/j.snb.2024.135530 SN - 0925-4005 (Print) SN - 1873-3077 (Online) N1 - Corresponding Author: Michael J. Schöning VL - 408 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Yoshinobu, Tatsuo A1 - Miyamoto, Ko-ichiro A1 - Wagner, Torsten A1 - Schöning, Michael Josef T1 - Field-effect sensors combined with the scanned light pulse technique: from artificial olfactory images to chemical imaging technologies JF - Chemosensors N2 - The artificial olfactory image was proposed by Lundström et al. in 1991 as a new strategy for an electronic nose system which generated a two-dimensional mapping to be interpreted as a fingerprint of the detected gas species. The potential distribution generated by the catalytic metals integrated into a semiconductor field-effect structure was read as a photocurrent signal generated by scanning light pulses. The impact of the proposed technology spread beyond gas sensing, inspiring the development of various imaging modalities based on the light addressing of field-effect structures to obtain spatial maps of pH distribution, ions, molecules, and impedance, and these modalities have been applied in both biological and non-biological systems. These light-addressing technologies have been further developed to realize the position control of a faradaic current on the electrode surface for localized electrochemical reactions and amperometric measurements, as well as the actuation of liquids in microfluidic devices. KW - visualization KW - light-addressing technologies KW - scanned light pulse technique KW - field-effect structure KW - MOS KW - metal-oxide-semiconductor structure KW - catalytic metal KW - electronic nose KW - gas sensor KW - artificial olfactory image Y1 - 2024 U6 - http://dx.doi.org/10.3390/chemosensors12020020 SN - 2227-9040 N1 - This article belongs to the Special Issue "An Exciting Journey of Chemical Sensors and Biosensors: A Theme Issue in Honor of Professor Ingemar Lundström" Corresponding author: Tatsuo Yoshinobu, Michael J. Schöning VL - 12 IS - 2 PB - MDPI CY - Basel ER - TY - JOUR A1 - Bertz, Morten A1 - Schöning, Michael Josef A1 - Molinnus, Denise A1 - Homma, Takayuki T1 - Influence of temperature, light, and H₂O₂ concentration on microbial spore inactivation: in-situ Raman spectroscopy combined with optical trapping JF - Physica status solidi (a) applications and materials science N2 - To gain insight on chemical sterilization processes, the influence of temperature (up to 70 °C), intense green light, and hydrogen peroxide (H₂O₂) concentration (up to 30% in aqueous solution) on microbial spore inactivation is evaluated by in-situ Raman spectroscopy with an optical trap. Bacillus atrophaeus is utilized as a model organism. Individual spores are isolated and their chemical makeup is monitored under dynamically changing conditions (temperature, light, and H₂O₂ concentration) to mimic industrially relevant process parameters for sterilization in the field of aseptic food processing. While isolated spores in water are highly stable, even at elevated temperatures of 70 °C, exposure to H₂O₂ leads to a loss of spore integrity characterized by the release of the key spore biomarker dipicolinic acid (DPA) in a concentration-dependent manner, which indicates damage to the inner membrane of the spore. Intensive light or heat, both of which accelerate the decomposition of H₂O₂ into reactive oxygen species (ROS), drastically shorten the spore lifetime, suggesting the formation of ROS as a rate-limiting step during sterilization. It is concluded that Raman spectroscopy can deliver mechanistic insight into the mode of action of H₂O₂-based sterilization and reveal the individual contributions of different sterilization methods acting in tandem. KW - hydrogen peroxide KW - optical spore trapping KW - Raman spectroscopy KW - sterilization conditions KW - temperature Y1 - 2024 U6 - http://dx.doi.org/10.1002/pssa.202300866 SN - 1862-6319 (Online) SN - 1862-6300 (Print) IS - Early View PB - Wiley-VCH CY - Berlin ER - TY - JOUR A1 - Engelmann, Ulrich M. A1 - Simsek, Beril A1 - Shalaby, Ahmed A1 - Krause, Hans-Joachim T1 - Key contributors to signal generation in frequency mixing magnetic detection (FMMD): an in silico study JF - Sensors N2 - Frequency mixing magnetic detection (FMMD) is a sensitive and selective technique to detect magnetic nanoparticles (MNPs) serving as probes for binding biological targets. Its principle relies on the nonlinear magnetic relaxation dynamics of a particle ensemble interacting with a dual frequency external magnetic field. In order to increase its sensitivity, lower its limit of detection and overall improve its applicability in biosensing, matching combinations of external field parameters and internal particle properties are being sought to advance FMMD. In this study, we systematically probe the aforementioned interaction with coupled Néel–Brownian dynamic relaxation simulations to examine how key MNP properties as well as applied field parameters affect the frequency mixing signal generation. It is found that the core size of MNPs dominates their nonlinear magnetic response, with the strongest contributions from the largest particles. The drive field amplitude dominates the shape of the field-dependent response, whereas effective anisotropy and hydrodynamic size of the particles only weakly influence the signal generation in FMMD. For tailoring the MNP properties and parameters of the setup towards optimal FMMD signal generation, our findings suggest choosing large particles of core sizes dc > 25 nm nm with narrow size distributions (σ < 0.1) to minimize the required drive field amplitude. This allows potential improvements of FMMD as a stand-alone application, as well as advances in magnetic particle imaging, hyperthermia and magnetic immunoassays. KW - key performance indicators KW - magnetic biosensing KW - coupled Néel–Brownian relaxation dynamics KW - frequency mixing magnetic detection KW - magnetic relaxation KW - micromagnetic simulation KW - magnetic nanoparticles Y1 - 2024 U6 - http://dx.doi.org/10.3390/s24061945 SN - 1424-8220 N1 - This article belongs to the Special Issue "Advances in Magnetic Sensors and Their Applications" VL - 24 IS - 6 PB - MDPI CY - Basel ER - TY - JOUR A1 - Haeger, Gerrit A1 - Jolmes, Tristan A1 - Oyen, Sven A1 - Jaeger, Karl-Erich A1 - Bongaerts, Johannes A1 - Schörken, Ulrich A1 - Siegert, Petra T1 - Novel recombinant aminoacylase from Paraburkholderia monticola capable of N-acyl-amino acid synthesis JF - Applied Microbiology and Biotechnology N2 - 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. KW - Chaperone KW - Biocatalysis KW - Aminoacylase KW - Acylation KW - Acyl-amino acids KW - Biosurfactants Y1 - 2024 U6 - http://dx.doi.org/10.1007/s00253-023-12868-8 SN - 1432-0614 N1 - Corresponding author: Petra Siegert IS - 108 PB - Springer CY - Berlin ER - TY - JOUR A1 - Karschuck, Tobias A1 - Schmidt, Stefan A1 - Achtsnicht, Stefan A1 - Poghossian, Arshak A1 - Wagner, Patrick A1 - Schöning, Michael Josef T1 - Multiplexing system for automated characterization of a capacitive field-effect sensor array JF - Physica Status Solidi A N2 - In comparison to single-analyte devices, multiplexed systems for a multianalyte detection offer a reduced assay time and sample volume, low cost, and high throughput. Herein, a multiplexing platform for an automated quasi-simultaneous characterization of multiple (up to 16) capacitive field-effect sensors by the capacitive–voltage (C–V) and the constant-capacitance (ConCap) mode is presented. The sensors are mounted in a newly designed multicell arrangement with one common reference electrode and are electrically connected to the impedance analyzer via the base station. A Python script for the automated characterization of the sensors executes the user-defined measurement protocol. The developed multiplexing system is tested for pH measurements and the label-free detection of ligand-stabilized, charged gold nanoparticles. KW - Capacitive field-effect sensor KW - Gold nanoparticles KW - Label-free detection KW - Multicell KW - Multiplexing Y1 - 2023 U6 - http://dx.doi.org/10.1002/pssa.202300265 SN - 1862-6300 (Print) SN - 1862-6319 (Online) N1 - Corresponding author: Michael Josef Schöning VL - 220 IS - 22 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Janus, Kevin Alexander A1 - Achtsnicht, Stefan A1 - Drinic, Aleksander A1 - Kopp, Alexander A1 - Keusgen, Michael A1 - Schöning, Michael Josef T1 - Transient magnesium-based thin-film temperature sensor on a flexible, bioabsorbable substrate for future medical applications JF - Applied Research N2 - In this work, the bioabsorbable materials, namely fibroin, polylactide acid (PLA), magnesium and magnesium oxide are investigated for their application as transient, resistive temperature detectors (RTD). For this purpose, a thin-film magnesium-based meander-like electrode is deposited onto a flexible, bioabsorbable substrate (fibroin or PLA) and encapsulated (passivated) by additional magnesium oxide layers on top and below the magnesium-based electrode. The morphology of different layered RTDs is analyzed by scanning electron microscopy. The sensor performance and lifetime of the RTD is characterized both under ambient atmospheric conditions between 30°C and 43°C, and wet tissue-like conditions with a constant temperature regime of 37°C. The latter triggers the degradation process of the magnesium-based layers. The 3-layers RTDs on a PLA substrate could achieve a lifetime of 8.5 h. These sensors also show the best sensor performance under ambient atmospheric conditions with a mean sensitivity of 0.48 Ω/°C ± 0.01 Ω/°C. KW - Silk fibroin KW - Polylactide acid KW - Bioabsorbable KW - Resistive temperature detector Y1 - 2023 U6 - http://dx.doi.org/10.1002/appl.202300102 SN - 2702-4288 (Print) N1 - Corresponding author: Michael Josef Schöning IS - Accepted manuscript PB - Wiley-VCH ER - TY - JOUR A1 - Haeger, Gerrit A1 - Probst, Johanna A1 - Jaeger, Karl-Erich A1 - Bongaerts, Johannes A1 - Siegert, Petra T1 - Novel aminoacylases from Streptomyces griseus DSM 40236 and their recombinant production in Streptomyces lividans JF - FEBS Open Bio N2 - Amino acid-based surfactants are valuable compounds for cosmetic formulations. The chemical synthesis of acyl-amino acids is conventionally performed by the Schotten-Baumann reaction using fatty acyl chlorides, but aminoacylases have also been investigated for use in biocatalytic synthesis with free fatty acids. Aminoacylases and their properties are diverse; they belong to different peptidase families and show differences in substrate specificity and biocatalytic potential. Bacterial aminoacylases capable of synthesis have been isolated from Burkholderia, Mycolicibacterium, and Streptomyces. Although several proteases and peptidases from S. griseus have been described, no aminoacylases from this species have been identified yet. In this study, we investigated two novel enzymes produced by S. griseus DSM 40236ᵀ . We identified and cloned the respective genes and recombinantly expressed an α-aminoacylase (EC 3.5.1.14), designated SgAA, and an ε-lysine acylase (EC 3.5.1.17), designated SgELA, in S. lividans TK23. The purified aminoacylase SgAA was biochemically characterized, focusing on its hydrolytic activity to determine temperature- and pH optima and stabilities. The aminoacylase could hydrolyze various acetyl-amino acids at the Nα -position with a broad specificity regarding the sidechain. Substrates with longer acyl chains, like lauroyl-amino acids, were hydrolyzed to a lesser extent. Purified aminoacylase SgELA specific for the hydrolysis of Nε -acetyl-L-lysine was unstable and lost its enzymatic activity upon storage for a longer period but could initially be characterized. The pH optimum of SgELA was pH 8.0. While synthesis of acyl-amino acids was not observed with SgELA, SgAA catalyzed the synthesis of lauroyl-methionine. KW - Streptomyces lividans KW - recombinant expression KW - Streptomyces griseus KW - ε-lysine acylase KW - α-aminoacylase Y1 - 2023 U6 - http://dx.doi.org/10.1002/2211-5463.13723 SN - 2211-5463 N1 - Corresponding author: Petra Siegert VL - 13 IS - 12 SP - 2224 EP - 2238 PB - Wiley CY - Hoboken, NJ ER - TY - JOUR A1 - Wendlandt, Tim A1 - Koch, Claudia A1 - Britz, Beate A1 - Liedek, Anke A1 - Schmidt, Nora A1 - Werner, Stefan A1 - Gleba, Yuri A1 - Vahidpour, Farnoosh A1 - Welden, Melanie A1 - Poghossian, Arshak A1 - Schöning, Michael Josef T1 - Facile Purification and Use of Tobamoviral Nanocarriers for Antibody-Mediated Display of a Two-Enzyme System JF - Viruses N2 - Immunosorbent turnip vein clearing virus (TVCV) particles displaying the IgG-binding domains D and E of Staphylococcus aureus protein A (PA) on every coat protein (CP) subunit (TVCVPA) were purified from plants via optimized and new protocols. The latter used polyethylene glycol (PEG) raw precipitates, from which virions were selectively re-solubilized in reverse PEG concentration gradients. This procedure improved the integrity of both TVCVPA and the wild-type subgroup 3 tobamovirus. TVCVPA could be loaded with more than 500 IgGs per virion, which mediated the immunocapture of fluorescent dyes, GFP, and active enzymes. Bi-enzyme ensembles of cooperating glucose oxidase and horseradish peroxidase were tethered together on the TVCVPA carriers via a single antibody type, with one enzyme conjugated chemically to its Fc region, and the other one bound as a target, yielding synthetic multi-enzyme complexes. In microtiter plates, the TVCVPA-displayed sugar-sensing system possessed a considerably increased reusability upon repeated testing, compared to the IgG-bound enzyme pair in the absence of the virus. A high coverage of the viral adapters was also achieved on Ta2O5 sensor chip surfaces coated with a polyelectrolyte interlayer, as a prerequisite for durable TVCVPA-assisted electrochemical biosensing via modularly IgG-assembled sensor enzymes. KW - biosensor KW - horseradish peroxidase (HRP) KW - glucose oxidase (GOx) KW - enzyme cascade KW - turnip vein clearing virus (TVCV) KW - tobacco mosaic virus (TMV) Y1 - 2023 U6 - http://dx.doi.org/doi.org/10.3390/v15091951 SN - 1999-4915 N1 - This article belongs to the Special Issue "Tobamoviruses 2023" VL - 9 IS - 15 PB - MDPI CY - Basel ER - TY - JOUR A1 - Bertz, Morten A1 - Molinnus, Denise A1 - Schöning, Michael Josef A1 - Homma, Takayuki T1 - Real-time monitoring of H₂O₂ sterilization on individual bacillus atrophaeus spores by optical sensing with trapping Raman spectroscopy JF - Chemosensors N2 - Hydrogen peroxide (H₂O₂), a strong oxidizer, is a commonly used sterilization agent employed during aseptic food processing and medical applications. To assess the sterilization efficiency with H₂O₂, bacterial spores are common microbial systems due to their remarkable robustness against a wide variety of decontamination strategies. Despite their widespread use, there is, however, only little information about the detailed time-resolved mechanism underlying the oxidative spore death by H₂O₂. In this work, we investigate chemical and morphological changes of individual Bacillus atrophaeus spores undergoing oxidative damage using optical sensing with trapping Raman microscopy in real-time. The time-resolved experiments reveal that spore death involves two distinct phases: (i) an initial phase dominated by the fast release of dipicolinic acid (DPA), a major spore biomarker, which indicates the rupture of the spore’s core; and (ii) the oxidation of the remaining spore material resulting in the subsequent fragmentation of the spores’ coat. Simultaneous observation of the spore morphology by optical microscopy corroborates these mechanisms. The dependence of the onset of DPA release and the time constant of spore fragmentation on H₂O₂ shows that the formation of reactive oxygen species from H₂O₂ is the rate-limiting factor of oxidative spore death. KW - DPA (dipicolinic acid) KW - sterilization KW - Bacillus atrophaeus spores KW - optical trapping KW - Raman spectroscopy KW - optical sensor setup Y1 - 2023 U6 - http://dx.doi.org/10.3390/chemosensors11080445 SN - 2227-9040 N1 - This article belongs to the Special Issue "Biosensors and Chemical Sensors for Food and Healthcare Monitoring—Celebrating the 10th Anniversary" VL - 8 IS - 11 PB - MDPI CY - Basel ER - TY - JOUR A1 - Falkenberg, Fabian A1 - Kohn, Sophie A1 - Bott, Michael A1 - Bongaerts, Johannes A1 - Siegert, Petra T1 - Biochemical characterisation of a novel broad pH spectrum subtilisin from Fictibacillus arsenicus DSM 15822ᵀ JF - FEBS Open Bio N2 - Subtilisins from microbial sources, especially from the Bacillaceae family, are of particular interest for biotechnological applications and serve the currently growing enzyme market as efficient and novel biocatalysts. Biotechnological applications include use in detergents, cosmetics, leather processing, wastewater treatment and pharmaceuticals. To identify a possible candidate for the enzyme market, here we cloned the gene of the subtilisin SPFA from Fictibacillus arsenicus DSM 15822ᵀ (obtained through a data mining-based search) and expressed it in Bacillus subtilis DB104. After production and purification, the protease showed a molecular mass of 27.57 kDa and a pI of 5.8. SPFA displayed hydrolytic activity at a temperature optimum of 80 °C and a very broad pH optimum between 8.5 and 11.5, with high activity up to pH 12.5. SPFA displayed no NaCl dependence but a high NaCl tolerance, with decreasing activity up to concentrations of 5 m NaCl. The stability enhanced with increasing NaCl concentration. Based on its substrate preference for 10 synthetic peptide 4-nitroanilide substrates with three or four amino acids and its phylogenetic classification, SPFA can be assigned to the subgroup of true subtilisins. Moreover, SPFA exhibited high tolerance to 5% (w/v) SDS and 5% H₂O₂ (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H₂O₂, suggest it has potential for biotechnological applications. KW - Bacillaceae KW - Biotechnological application KW - Broad pH spectrum KW - Subtilases KW - Subtilisin Y1 - 2023 U6 - http://dx.doi.org/10.1002/2211-5463.13701 SN - 2211-5463 N1 - Corresponding author: Petra Siegert VL - 13 IS - 11 SP - 2035 EP - 2046 PB - Wiley CY - Hoboken, NJ ER - TY - JOUR A1 - Morais, Paulo V. A1 - Suman, Pedro H. A1 - Schöning, Michael Josef A1 - Siqueira Junior, José R. A1 - Orlandi, Marcelo O. T1 - Layer-by-layer film based on Sn₃O₄ nanobelts as sensing units to detect heavy metals using a capacitive field-effect sensor platform JF - Chemosensors N2 - Lead and nickel, as heavy metals, are still used in industrial processes, and are classified as “environmental health hazards” due to their toxicity and polluting potential. The detection of heavy metals can prevent environmental pollution at toxic levels that are critical to human health. In this sense, the electrolyte–insulator–semiconductor (EIS) field-effect sensor is an attractive sensing platform concerning the fabrication of reusable and robust sensors to detect such substances. This study is aimed to fabricate a sensing unit on an EIS device based on Sn₃O₄ nanobelts embedded in a polyelectrolyte matrix of polyvinylpyrrolidone (PVP) and polyacrylic acid (PAA) using the layer-by-layer (LbL) technique. The EIS-Sn₃O₄ sensor exhibited enhanced electrochemical performance for detecting Pb²⁺ and Ni²⁺ ions, revealing a higher affinity for Pb²⁺ ions, with sensitivities of ca. 25.8 mV/decade and 2.4 mV/decade, respectively. Such results indicate that Sn₃O₄ nanobelts can contemplate a feasible proof-of-concept capacitive field-effect sensor for heavy metal detection, envisaging other future studies focusing on environmental monitoring. KW - Sn₃O₄ KW - nanobelts KW - field-effect sensor KW - LbL films KW - heavy metals Y1 - 2023 U6 - http://dx.doi.org/10.3390/chemosensors11080436 SN - 2227-9040 N1 - This article belongs to the Special Issue The Application of Electrochemical Sensors or Biosensors Based on Nanomaterials VL - 11 IS - 8 PB - MDPI CY - Basel ER - TY - RPRT A1 - Siegert, Petra A1 - Bongaerts, Johannes A1 - Wagner, Torsten A1 - Schöning, Michael Josef A1 - Selmer, Thorsten T1 - Abschlussbericht zum Projekt zur Überwachung biotechnologischer Prozesse mittels Diacetyl-/Acetoin-Biosensor und Evaluierung von Acetoin-Reduktasen zur Verwendung in Biotransformationen Y1 - 2022 N1 - Laufzeit: 01.01.2016 – 31.12.2019 (verlängert bis 31.12.2020) Förderkennzeichen: 322-8.03.04.02-FH-Struktur 2016/02 Gefördert durch: Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen CY - Aachen ER - TY - RPRT A1 - Haeger, Gerrit A1 - Bongaerts, Johannes A1 - Siegert, Petra T1 - Abschlussbericht Teil II: Eingehende Darstellung Neue biobasierte Lipopeptide aus nachhaltiger Produktion (LipoPep) Y1 - 2023 N1 - Förderkennzeichen: 13FH256PA6 Titel: FHprofUnt 2016: Neue biobasierte Lipopeptide aus nachhaltiger Produktion Laufzeit: 01.02.2019 – 31.10.2022 ER - TY - JOUR A1 - Rabehi, Amine A1 - Garlan, Benjamin A1 - Achtsnicht, Stefan A1 - Krause, Hans-Joachim A1 - Offenhäusser, Andreas A1 - Ngo, Kieu A1 - Neveu, Sophie A1 - Graff-Dubois, Stephanie A1 - Kokabi, Hamid T1 - Magnetic detection structure for Lab-on-Chip applications based on the frequency mixing technique JF - Sensors N2 - 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. KW - Lab-on-Chip KW - magnetic sensing KW - frequency mixing KW - superparamagnetic nanoparticles KW - magnetic beads Y1 - 2018 U6 - http://dx.doi.org/10.3390/s18061747 SN - 1424-8220 VL - 18 IS - 6 PB - MDPI CY - Basel ER - TY - JOUR A1 - Achtsnicht, Stefan A1 - Schönenborn, Kristina A1 - Offenhäusser, Andreas A1 - Krause, Hans-Joachim T1 - Measurement of the magnetophoretic velocity of different superparamagnetic beads JF - Journal of Magnetism and Magnetic Materials N2 - 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. KW - magnetophoretic velocity KW - superparamagnetic bead KW - magnetic tweezers KW - magnetic separation KW - magnetic actuation Y1 - 2019 U6 - http://dx.doi.org/10.1016/j.jmmm.2018.10.066 SN - 0304-8853 VL - 477 IS - 1 SP - 244 EP - 248 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Achtsnicht, Stefan A1 - Tödter, Julia A1 - Niehues, Julia A1 - Telöken, Matthias A1 - Offenhäusser, Andreas A1 - Krause, Hans-Joachim A1 - Schröper, Florian T1 - 3D printed modular immunofiltration columns for frequency mixing-based multiplex magnetic immunodetection JF - Sensors N2 - 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. Y1 - 2019 U6 - http://dx.doi.org/10.3390/s19010148 SN - 1424-8220 VL - 19 IS - 1 PB - MDPI CY - Basel ER - TY - JOUR A1 - Achtsnicht, Stefan A1 - Pourshahidi, Ali Mohammad A1 - Offenhäusser, Andreas A1 - Krause, Hans-Joachim T1 - Multiplex detection of different magnetic beads using frequency scanning in magnetic frequency mixing technique JF - Sensors N2 - 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. KW - frequency mixing magnetic detection KW - magnetic sandwich immunoassay KW - multiparametric immunoassays Y1 - 2019 U6 - http://dx.doi.org/10.3390/s19112599 SN - 1424-8220 VL - 19 IS - 11 PB - MDPI CY - Basel ER - TY - JOUR A1 - Achtsnicht, Stefan A1 - Neuendorf, Christian A1 - Faßbender, Tobias A1 - Nölke, Greta A1 - Offenhäusser, Andreas A1 - Krause, Hans-Joachim A1 - Schröper, Florian T1 - Sensitive and rapid detection of cholera toxin subunit B using magnetic frequency mixing detection JF - Plos One N2 - 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. Y1 - 2019 U6 - http://dx.doi.org/10.1371/journal.pone.0219356 SN - 1932-6203 VL - 14 IS - 7 PB - Plos CY - San Francisco ER - TY - THES A1 - Achtsnicht, Stefan T1 - Multiplex-Magnetdetektion von superparamagnetischen Beads zur Identifizierung von Trinkwasserkontaminationen N2 - Die qualitative und quantitative Detektion von Zielsubstanzen innerhalb einer wässrigen Probe ist für viele Fragestellungen von Interesse, etwa bei der Detektion von Kontaminationen in Trinkwasser in Krisensituationen. Hierbei ist es nicht nur wichtig, dass Pathogene möglichst sensitiv detektiert werden können, sondern auch, dass die Analyse schnell erfolgt, um Betroffenen im Katastrophenfall zügig sicheres Trinkwasser zu Verfügung stellen zu können. Da bei einem solchen Szenario nicht von einer in der Nähe 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 möglich ist. Dabei werden die MBs mit Hilfe von primären Antikörpern an die Zielsubstanz gebunden und mit sekundären Antikörpern an die Poren-Oberfläche eines Polyethylen-Filters fixiert (Sandwich-Immunoassay). So kann die Quantifizierung der Zielsubstanz auf eine magnetische Messung der immobilisierten MB-Marker zurückgeführt 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 Handheldgerät realisiert wurde. Zusätzlich 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 Möglichkeit zur Sensitivitätssteigerung ist die magnetische Aufkonzentration, indem vor der magnetischen Analyse eine Separation der MBs aus einem größeren Probenvolumen mittels magnetischem Feldgradienten durchgeführt 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 Ansätze entwickelt und getestet, die einen solchen multiparametrischen magnetischen Immunoassay ermöglichen. Einerseits wurde eine räumliche Separation der Bindungsbereiche für verschiedene Zielsubstanzen realisiert, die sequentiell ausgewertet werden können. Andererseits wurde die Unterscheidung von verschiedenen Zielsubstanzen anhand der Charakteristika der an sie gebundenen, verschieden funktionalisierten MB-Typen untersucht. Für eine solche Unterscheidung wurde zum einen die Anregefrequenz der magnetischen Frequenzmischtechnik während 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 binären Mischung zweier verschiedener MB-Typen als gradueller Übergang der Verläufe der beiden reinen MB-Lösungen ergibt. Eine weitere Analysemethode für einen multiparametrischen Immunoassay besteht darin, ein zusätzliches einstellbares statisches magnetisches Offsetfeld zu verwenden. Hierfür 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 für MBs mit unterschiedlichen magnetischen Partikelmomenten möglich 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. N2 - The qualitative and quantitative detection of target substances in an aqueous sample is of interest for many questions, for example in the detection of contaminations in drinking water in crisis situations. It is not only important that pathogens can be detected with highest possible sensitivity, but also that the analysis is carried out quickly so that safe drinking water can be provided in the event of a disaster. During such a scenario one cannot rely on a functioning laboratory infrastructure nearby. Therefore it is important that the measurement can be carried out directly on site. Within the scope of this work, it was investigated whether such a quick analysis can be performed using superparamagnetic beads (MBs) and the magnetic frequency mixing technique. The MBs are bound to the target substance with the aid of primary antibodies and fixed to the pore surfaces of a polyethylene filter with secondary antibodies (sandwich immunoassay). The quantification of the target substance can thus be traced back to a magnetic measurement of the immobilized MB markers. The magnetic frequency mixing technique is based on the excitation of the sample with magnetic fields of two different frequencies. The mixing frequencies generated due to the non-linear shape of the magnetization of the superparamagnetic MBs are typically analyzed using a two-stage Lock-in detection (analog demodulation), which was implemented in a magnetic reader as a handheld device. In addition to this technique, the principle of direct digitization of the entire response signal with subsequent Fourier analysis of the generated mixing frequencies was experimentally implemented in order to simultaneously record the amplitudes and phases of several mixing frequencies. One possibility for increasing the sensitivity is magnetic concentration. In that case, the MBs are separated from a larger sample volume by means of a magnetic field gradient before the magnetic analysis. To characterize various commercial MBs with regard to their magnetic separability, a setup for measuring their magnetophoretic mobility was implemented and their velocities in the gradient field were measured with an optical microscope.Often, a sample has to be examined not only for a single target substance, but for several different pathogens simultaneously. Various approaches have been developed and tested which enable such a multiparametric magnetic immunoassay. On the one hand, a spatial separation of the binding areas for different target substances was realized, which can be evaluated sequentially. On the other hand, a distinction among different target substances based on the magnetic characteristics of their attached different MB types was examined. For this discrimination, the excitation frequency of the magnetic frequency mixing technology was varied during measurement. It is shown that different MB types can be distinguished from one another based on the phase of their frequency mixing signals. The signal curve of a binary mixture of two different MB types is obtained as a gradual transition of the curves of the two pure MB solutions. Another method of analysis for a multiparametric immunoassay is based on an additional adjustable static magnetic offset field. For this purpose, several setups based on permanent magnets and electromagnets were simulated, designed and characterized. The simulations show that a distinction based on this method is possible for MBs with different magnetic particle moments. As a direct application of the developed magnetic reader in conjunction with digital demodulation, a magnetic assay against the B subunit of cholera toxin in drinking water was demonstrated, and a low detection limit of 0.2 ng/ml was achieved. KW - Choleratoxin B KW - Trinkwassersicherheit KW - cholera toxin B KW - drinking water safety KW - magnetic frequency mixing technique Y1 - 2020 U6 - http://dx.doi.org/10.18154/RWTH-2020-12052 N1 - Dissertation, RWTH Aachen University, 2020 ER -