Browsing by Author "Wendlandt, Tim"

Now showing 1 - 4 of 4

Open Access
Facile purification and use of tobamoviral nanocarriers for antibody-mediated display of a two-enzyme system
(2023) Wendlandt, Tim; Koch, Claudia; Britz, Beate; Liedek, Anke; Schmidt, Nora; Werner, Stefan; Gleba, Yuri; Vahidpour, Farnoosh; Welden, Melanie; Poghossian, Arshak; Schöning, Michael J.; Eber, Fabian J.; Jeske, Holger; Wege, Christina
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.
Open Access
Getting hold of the tobamovirus particle : why and how? Purification routes over time and a new customizable approach
(2024) Wendlandt, Tim; Britz, Beate; Kleinow, Tatjana; Hipp, Katharina; Eber, Fabian J.; Wege, Christina
This article develops a multi-perspective view on motivations and methods for tobamovirus purification through the ages and presents a novel, efficient, easy-to-use approach that can be well-adapted to different species of native and functionalized virions. We survey the various driving forces prompting researchers to enrich tobamoviruses, from the search for the causative agents of mosaic diseases in plants to their increasing recognition as versatile nanocarriers in biomedical and engineering applications. The best practices and rarely applied options for the serial processing steps required for successful isolation of tobamoviruses are then reviewed. Adaptations for distinct particle species, pitfalls, and ‘forgotten’ or underrepresented technologies are considered as well. The article is topped off with our own development of a method for virion preparation, rooted in historical protocols. It combines selective re-solubilization of polyethylene glycol (PEG) virion raw precipitates with density step gradient centrifugation in biocompatible iodixanol formulations, yielding ready-to-use particle suspensions. This newly established protocol and some considerations for perhaps worthwhile further developments could serve as putative stepping stones towards preparation procedures appropriate for routine practical uses of these multivalent soft-matter nanorods.
Open Access
Soft sub‐structured multi‐material biosensor hydrogels with enzymes retained by plant viral scaffolds
(2023) Grübel, Jana; Wendlandt, Tim; Urban, Daniela; Jauch, Corinna O.; Wege, Christina; Tovar, Günter E. M.; Southan, Alexander
An all‐soft multi‐material combination consisting of a hydrogel based on poly(ethylene glycol) (PEG) coated with spatially defined spots of gelatin methacryloyl (GM) containing selectively addressable viral nanorods is presented, and its basic application as a qualitative biosensor with reporter enzymes displayed on the tobacco mosaic virus (TMV) bioscaffolds within the GM is demonstrated. Biologically inert PEG supports are equipped with GM spots serving as biological matrix for enzymes clustered on TMV particles preventing diffusion out of the gel. For this multi‐material combination, i) the PEG‐based hydrogel surface is modified to achieve a clear boundary between coated and non‐coated regions by introducing either isothiouronium or thiol groups. ii) Cross‐linking of the GM spots is studied to achieve anchoring to the hydrogel surface. iii) The enzymes horseradish peroxidase or penicillinase (Pen) are conjugated to TMV and integrated into the GM matrix. In contrast to free enzymes, enzyme‐decorated TMVs persist in GM spots and show sustained enzyme activity as evidenced by specific color reaction after 7 days of washing, and for Pen after 22 months after dry storage. Therefore, the integration of enzyme‐coupled TMV into hydrogel matrices is a promising and versatile approach to obtaining reusable and analyte‐specific sensor components.
Open Access
Towards multi-analyte detection with field-effect capacitors modified with tobacco mosaic virus bioparticles as enzyme nanocarriers
(2022) Welden, Melanie; Poghossian, Arshak; Vahidpour, Farnoosh; Wendlandt, Tim; Keusgen, Michael; Wege, Christina; Schöning, Michael J.
Utilizing an appropriate enzyme immobilization strategy is crucial for designing enzyme-based biosensors. Plant virus-like particles represent ideal nanoscaffolds for an extremely dense and precise immobilization of enzymes, due to their regular shape, high surface-to-volume ratio and high density of surface binding sites. In the present work, tobacco mosaic virus (TMV) particles were applied for the co-immobilization of penicillinase and urease onto the gate surface of a field-effect electrolyte-insulator-semiconductor capacitor (EISCAP) with a p-Si-SiO2-Ta2O5 layer structure for the sequential detection of penicillin and urea. The TMV-assisted bi-enzyme EISCAP biosensor exhibited a high urea and penicillin sensitivity of 54 and 85 mV/dec, respectively, in the concentration range of 0.1–3 mM. For comparison, the characteristics of single-enzyme EISCAP biosensors modified with TMV particles immobilized with either penicillinase or urease were also investigated. The surface morphology of the TMV-modified Ta2O5-gate was analyzed by scanning electron microscopy. Additionally, the bi-enzyme EISCAP was applied to mimic an XOR (Exclusive OR) enzyme logic gate.