TY - JOUR A1 - Dürig, Wiebke A1 - Lindblad, Sofia A1 - Golovko, Oksana A1 - Gkotsis, Georgios A1 - Aalizadeh, Reza A1 - Nika, Maria-Christina A1 - Thomaidis, Nikolaos A1 - Alygizakis, Nikiforos A. A1 - Plassmann, Merle A1 - Haglund, Peter A1 - Fu, Qiuguo A1 - Hollender, Juliane A1 - Chaker, Jade A1 - David, Arthur A1 - Kunkel, Uwe A1 - Macherius, André A1 - Belova, Lidia A1 - Poma, Giulia A1 - Preud'Homme, Hugues A1 - Munschy, Catherine A1 - Aminot, Yann A1 - Jaeger, Carsten A1 - Lisec, Jan A1 - Hansen, Martin A1 - Vorkamp, Katrin A1 - Zhu, Linyan A1 - Cappelli, Francesca A1 - Roscioli, Claudio A1 - Valsecchi, Sara A1 - Bagnati, Renzo A1 - González, Belén A1 - Prieto, Ailette A1 - Zuloaga, Olatz A1 - Gil-Solsona, Ruben A1 - Gago-Ferrero, Pablo A1 - Rodriguez-Mozaz, Sara A1 - Budzinski, Hélène A1 - Devier, Marie-Helene A1 - Dierkes, Georg A1 - Boulard, Lise A1 - Jacobs, Griet A1 - Voorspoels, Stefan A1 - Rüdel, Heinz A1 - Ahrens, Lutz T1 - What is in the fish? Collaborative trial in suspect and non-target screening of organic micropollutants using LC- and GC-HRMS N2 - A collaborative trial involving 16 participants from nine European countries was conducted within the NORMAN network in efforts to harmonise suspect and non-target screening of environmental contaminants in whole fish samples of bream (Abramis brama). Participants were provided with freeze-dried, homogenised fish samples from a contaminated and a reference site, extracts (spiked and non-spiked) and reference sample preparation protocols for liquid chromatography (LC) and gas chromatography (GC) coupled to high resolution mass spectrometry (HRMS). Participants extracted fish samples using their in-house sample preparation method and/or the protocol provided. Participants correctly identified 9-69% of spiked compounds using LC-HRMS and 20-60% of spiked compounds using GC-HRMS. From the contaminated site, suspect screening with participants’ own suspect lists led to putative identification of on average ~145 and ~20 unique features per participant using LC-HRMS and GC-HRMS, respectively, while non-target screening identified on average ~42 and ~56 unique features per participant using LC-HRMS and GC-HRMS, respectively. Within the same sub-group of sample preparation method, only a few features were identified by at least two participants in suspect screening (16 features using LC-HRMS, 0 features using GC-HRMS) and non-target screening (0 features using LC-HRMS, 2 features using GC-HRMS). The compounds identified had log octanol/water partition coefficient (KOW) values ranging from -9.9 to 16 and mass-to-charge ratio (m/z) of 68 to 761 (LC-HRMS and GC-HRMS). A significant linear trend was found between log KOW and m/z for the GC-HRMS data. Overall, these findings indicate that differences in screening results are mainly due to the data analysis workflows used by different participants. Further work is needed to harmonise the results obtained when applying suspect and non-target screening approaches to environmental biota samples. KW - General Environmental Science KW - Suspect and non-target analysis KW - Biota KW - Expobome KW - Collaborative trial PY - 2023 DO - https://doi.org/10.1016/j.envint.2023.108288 VL - 181 SP - 1 EP - 24 PB - Elsevier B.V. AN - OPUS4-58681 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Vogel, Christian T1 - Per- and Polyfluoroalkyl Substances (PFAS) in Sewage Sludge and Wastewater-based Fertilizers N2 - Per- and polyfluoroalkyl substances (PFAS) are chemicals which were developed to improve humanity’s quality of life. Due to their high chemical stability and resistance to degradation by heat or acids, PFAS were used in a variety of consumer products. The continuous use of PFAS in household products and the discharge of PFAS from industrial plants into the sewer system resulted in the contamination of effluents and sewage sludge from wastewater treatment plants (WWTPs) (Roesch et al. 2022). Since sewage sludge is often used as fertilizer, its application on agricultural soils has been observed as a significant entry path for PFAS into the environment, specifically in our food chain. In Germany the sewage sludge/biosolid application on agricultural land was banned with the amendment of the German Sewage Sludge Ordinance and by 2029 sewage sludge application will be totally prohibited. However, phosphorus (P) from sewage sludge should still be recycled in WWTPs of cities with a population larger than 50,000 residents. To produce high-quality P-fertilizers for a circular economy, PFAS and other pollutants (e.g. pesticides and pharmaceuticals) must be separated from sewage sludge. Due to the strong diversity of industrial PFAS usage it is not clear if a safe application of novel recycled P-fertilizers from WWTPs can be guaranteed. Therefore, we analyzed various sewage sludges and wastewater-based fertilizers. Sewage sludge (SL) samples from various WWTPs in Germany and Switzerland, six sewage sludge ashes (SSA) from Germany, six thermally treated SL and SSA samples with different additives (temperatures: 700-1050 °C), two pyrolyzed SL samples (temperature: 400 °C) and two struvite samples from Germany and Canada were analyzed. The goal was to quantify PFAS in sewage sludges and wastewater-based P-fertilizers with the sum parameter extractable organic fluorine (EOF) by combustion ion chromatography (CIC). The results were compared with data from classical LC-MS/MS target analysis as well as selected samples by HR-MS suspect screening. The EOF values of the SLs mainly range between 154 and 538 µg/kg except for one SL which showed an elevated EOF value of 7209 µg/kg due to high organofluorine contamination. For the SSA samples the EOF values were lower and values between LOQ (approx. 60 µg/kg) and 121 µg/kg could be detected. For the pyrolyzed SLs no EOF values above the LOQ were detected. Moreover, the two wastewater-based struvite fertilizers contain 96 and 112 µg/kg EOF, respectively. In contrast to the EOF values, the sum of PFAS target values were relatively low for all SLs. Additional applied PFAS HR-MS suspect screening aimed to tentatively identify PFAS that could contribute to the hitherto unknown part of the EOF value. The majority of the detected fluorinated compounds are legacy PFAS such as short- and long-chain perfluorocarboxylic acids (PFCA), perfluorosulfonic acids (PFSA), polyfluoroalkyl phosphate esters (PAPs) and perfluorophosphonic acids (PFPA). Moreover, fluorinated pesticides, pharmaceutical as well as aromatic compounds were also identified, which are all included in the EOF parameter. Our research revealed that the current PFAS limit of 100 µg/kg for the sum of PFOS + PFOA in the German Fertilizer Ordinance is no longer up to date. Since the number of known PFAS already exceeds 10,000, the ordinance limit should be updated accordingly. Recent regulations and restrictions on using long-chain PFAS (≥C8) have resulted in a significant shift in the industry towards (ultra-)short-chain alternatives, and other, partly unknown, emerging PFAS. Ultimately, also fluorinated pesticides and pharmaceuticals, which end up as ultrashort PFAS in the WWTPs, have to be considered as possible pollutants in fertilizers from wastewater, too. T2 - Dioxin Konferenz CY - Maastricht, Netherlands DA - 10.09.2023 KW - Combustion Ion Chromatography KW - Per- and Polyfluoroalkyl substances (PFAS) KW - Sewage sludge KW - Fertilizer PY - 2023 AN - OPUS4-58345 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Hantschke, Luisa Lamberta T1 - Determination of background values of PFAS in representative soil samples in Germany N2 - Per- and polyfluoroalkyl substances (PFAS) are a large and ever-growing class of chemicals that are widely used. Due to the strength of the C-F bond, they do not decompose but accumulate in the environment posing a risk for nature and humans alike. While the use of some PFAS, like perfluorooctanoic acid (PFOA) and perfluorooctanoic sulfonic acid (PFOS) is already regulated in the EU, new ‘precursor’ substances are used to replace them. However, these precursors may be equally harmful to the environment. In recent years, many ‘hot spot’ sites with high PFAS contaminations in the soils have been distinguished,but no German-wide background values have been determined yet. Knowing these background values is crucial to understand the degree of PFAS contamination, underpinning future regulatory decisions. 600 soil samples are freshly taken at different sites all over Germany. PFAS are extracted from all samples. The extracts are analysed using LC-MS/MS with 30 PFAS as target compounds. Eluates are produced and analysed using targeted LC-MS/MS. To determine the amount of precursor substances not included in the targeted list, total oxidisable precursor (TOP) assays are performed with different approaches. Additionally, 50 samples taken at the same locations in 2012 are analysed to determine temporal trends of PFAS contamination. The determination of total extractable organically bound fluorine (EOF) is planned for comparison. The aim of the project is to obtain a broad overview of the status and development of the PFAS contamination in soil in Germany. By the preparation and analysis of eluates, the leaching behaviour of the analytes and thereby their mobility in the environment is investigated, helping to understand the transport of PFAS from contaminated soil to water. The use of different TOP assay methods allows to compare their results and usability in routine analysis, thereby giving insights into the amount of PFAS contamination hidden to targeted analysis and helping inspire future analysis strategies. The future comparison of these results to EOF analysis may further underline this point. The large number of samples and the geographic distribution of the sampling is unprecedented in Germany and allows to determine high-risk sites and ‘hot spots’. In the future, the insights from the project may be used as a guide for the political regulation of uses of PFAS. T2 - ANAKON23 CY - Vienna, Austria DA - 11.04.2023 KW - PFAS KW - Soil contamination KW - Background values KW - BBodSChV PY - 2023 AN - OPUS4-57755 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Singh, Chandan A1 - Riedel, Soraya A1 - Konthur, Zoltán A1 - Hodoroaba, Vasile-Dan A1 - Radnik, Jörg A1 - Schenk, J. A. A1 - Schneider, Rudolf T1 - Functionalized Ti3C2Tx nanosheets based biosensor for point-of-care detection of SARS-CoV‑2 antigen N2 - MXenes are considered a promising class of two-dimensional materials with extraordinary physical and electrochemical properties. Distinguished features like high specific surface area and outstanding electrical conductivity make them suitable for electrochemical biosensing applications. Here, we report the development of a biosensor involving the functionalized MXene−titanium carbide nanosheets (Ti3C2Tx-NS) and monoclonal antibodies against the SARS-CoV-2 nucleocapsid protein (anti-SARS-CoV-2 mAb) to design a point-of-care device for detection of the SARS-CoV-2 nucleocapsid protein (SARS-CoV-2 NP) antigen. Few-layered titanium carbide nanosheets (denoted as FL-Ti3C2Tx-NS) have been synthesized using a single-step etching and delamination method and characterized using optical and electron microscopy techniques revealing the suitability for immunosensing applications. Binding studies revealed the excellent affinity between the biosensor and the SARS-CoV-2 NP. Electrochemical detection of SARS-CoV-2 NP is performed using differential pulse voltammetry and read by a smartphone-based user interface. The proposed FL-Ti3C2Tx-NS based biosensor offers the detection of SARS-CoV-2 NP with a limit of detection of 0.91 nM in a wide detection range in spiked saliva samples. Additionally, there is no cross-reactivity in the presence of potential interferants like SARS-CoV-2 spike glycoprotein and bovine serum albumin. These findings demonstrate the potential of MXenes in developing a rapid and reliable tool for SARS-CoV-2 NP detection. While we report the biosensing of SARS-CoV-2 NP, our system also paves the way for the detection of other SARS-CoV-2 antigens like spike protein or other biomolecules based on antigen−antibody interactions. KW - Antigen testing KW - Few-layered titanium carbide nanosheets KW - SARS-CoV-2 nucleocapsid protein KW - Label-free detection KW - Electrochemical immunosensor PY - 2023 DO - https://doi.org/10.1021/acsaenm.2c00118 SN - 2771-9545 N1 - Geburtsname von Riedel, Soraya: Höfs, S. - Birth name of Riedel, Soraya: Höfs, S. VL - 1 IS - 1 SP - 495 EP - 507 PB - American Chemical Society CY - Washington, DC AN - OPUS4-56931 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Völzke, Jule L. A1 - Smatty, Sarah A1 - Döring, Sarah A1 - Ewald, Shireen A1 - Oelze, Marcus A1 - Fratzke, Franziska A1 - Flemig, Sabine A1 - Konthur, Zoltán A1 - Weller, Michael G. T1 - Efficient Purification of Polyhistidine-Tagged Recombinant Proteins Using Functionalized Corundum Particles N2 - Immobilized metal affinity chromatography (IMAC) is a widely used technique for purifying polyhistidine-tagged recombinant proteins. However, it often has practical limitations that require complex optimizations and additional steps for purification. In this study, we introduce functionalized corundum particles as a novel, efficient, and economical method for purifying recombinant proteins in a column-free format. The corundum surface is modified with amino silane APTES, followed by EDTA dianhydride, and then loaded with nickel ions. We used the Kaiser test to monitor the modification process and ICP-MS to quantify the metal-binding capacity. To evaluate the system, we used His-tagged protein A/G (PAG) mixed with bovine serum albumin (BSA). The corundum particles exhibited a binding capacity of approximately 3 mg of protein per gram of corundum or 2.4 mg per 1 mL of corundum suspension. We also examined cytoplasm obtained from different E. coli strains as an example of a complex matrix. Varying the imidazole concentration in the loading and washing buffers showed that higher concentrations during loading improved purity. Even with sample volumes as large as one liter, we successfully isolated recombinant proteins down to a concentration of 1 µg/mL. We found higher purity levels with corundum when comparing the corundum material to standard Ni–NTA agarose beads. We successfully purified His6-MBP-mSA2, a fusion protein comprising monomeric streptavidin and maltose-binding protein, from E. coli cytoplasm, demonstrating the method's applicability. We also purified SARS-CoV-2-S-RBD-His8 expressed in human Expi293F cells, confirming its suitability for mammalian cell culture supernatants. The material cost of the nickel-loaded corundum material (without regeneration) is estimated to be less than 30 cents per gram of functionalized support or 10 cents per milligram of isolated protein. Another advantage of this system is the exceptional physical and chemical stability of corundum particles. Overall, we have demonstrated that this novel material offers an efficient, robust, and cost-effective purification platform for His-tagged proteins, even in challenging, complex matrices and large sample volumes with low product concentrations. This method has potential applications in both small laboratories and large-scale industrial settings. N2 - Die immobilisierte Metallaffinitätschromatographie (IMAC) ist eine weit verbreitete Technik zur Reinigung von rekombinanten Proteinen mit Polyhistidin-Markierung. Sie hat jedoch oft praktische Einschränkungen, die komplexe Optimierungen und zusätzliche Schritte für die Aufreinigung erfordern. In dieser Studie stellen wir funktionalisierte Korundpartikel als neuartige, effiziente und wirtschaftliche Methode zur Reinigung rekombinanter Proteine in einem säulenfreien Format vor. Die Korundoberfläche wird mit dem Aminosilan APTES und anschließend mit EDTA-Dianhydrid modifiziert und dann mit Nickelionen beladen. Wir haben den Kaiser-Test zur Überwachung des Modifizierungsprozesses und ICP-MS zur Quantifizierung der Metallbindungskapazität verwendet. Zur Charakterisierung des Systems verwendeten wir His-markiertes Protein A/G (PAG) in Kombination mit Rinderserumalbumin (BSA). Die Korundpartikel wiesen eine Bindungskapazität von etwa 3 mg Protein pro Gramm Korund oder 2,4 mg pro 1 ml Korundsuspension auf. Als Beispiel für eine komplexe Matrix untersuchten wir auch Zytoplasma, das aus verschiedenen E. coli-Stämmen gewonnen wurde. Die Variation der Imidazolkonzentration in den Lade- und Waschpuffern zeigte, dass höhere Konzentrationen beim Laden die Reinheit verbesserten. Selbst bei einem Probenvolumen von bis zu einem Liter konnten wir rekombinante Proteine von 1 µg/mL isolieren. Beim Vergleich des Korundmaterials mit Standard-Ni-NTA-Agarose-Beads stellten wir einen höheren Reinheitsgrad mit Korund fest. Wir reinigten erfolgreich His6-MBP-mSA2 aus E. coli-Zytoplasma, ein Fusionsprotein, das aus monomerem Streptavidin und Maltose-bindendem Protein besteht. Wir reinigten auch SARS-CoV-2-S-RBD-His8, das in humanen Expi293F-Zellen exprimiert wurde, und bestätigten damit die Eignung des Materials für Zellkulturüberstände von Säugetieren. Die Materialkosten für nickelbeladenen Korund (ohne Regenerierung) werden auf weniger als 30 Cent pro Gramm funktionalisierten Trägers oder 10 Cent pro Milligramm isolierten Proteins geschätzt. Ein weiterer Vorteil dieses Systems ist die außergewöhnliche physikalische und chemische Stabilität der Korundpartikel. Insgesamt haben wir gezeigt, dass dieses neuartige Material eine effiziente, robuste und kostengünstige Reinigungsplattform für His-markierte Proteine bietet, selbst bei schwierigen, komplexen Matrices und großen Probenmengen mit niedrigen Produktkonzentrationen. Diese Methode könnte sowohl in kleinen Labors als auch in der Großindustrie eingesetzt werden. KW - Aluminum oxide KW - Sapphire KW - Ethylenediaminetetraacetic acid KW - Nickel chelate KW - EDTAD KW - HexaHis-Tag KW - His6 KW - 6xHis KW - His8 KW - Bioseparation KW - IMAC purification KW - Immunocapture KW - Affinity chromatography KW - Carrier KW - Nickel KW - Recombinant protein KW - Escherichia coli KW - Bacterial lysates KW - Cytoplasm KW - Polishing KW - Downstream processing PY - 2023 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-575672 DO - https://doi.org/10.3390/biotech12020031 VL - 12 IS - 2 SP - 1 EP - 18 PB - MDPI CY - Basel, Schweiz AN - OPUS4-57567 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Filimonova, S. M. A1 - Melnikov, E. S. A1 - Kaufmann, Jan Ole A1 - Shchepochkina, O. Y. A1 - Eremin, S. A. A1 - Gravel, I. V. A1 - Raysyan, Anna T1 - Exploring the anti‐α‐amylase activity of flavonoid aglycones in fabaceae plant extracts: a combined MALDI‐TOF‐MS and LC–MS/MS approach N2 - A combination of TLC-bioautography, MALDI-TOF-MS and LC–MS/MS methods was used to identify flavonoids with anti-α-amylase activity in extracts of Lathyrus pratensis L. (herb), L. polyphillus L. (fruits), Thermopsis lanceolata R. Br. (herb) and S. japonica L. (buds). After the TLC-autobiography assay, substances with anti-amylase activity were identified by MALDI-TOF-MS followed by confirmation of the result by LC–MS/MS. Results of the study revealed that the flavonoids apigenin, luteolin, formononetin, genistein and kaempferol display marked anti-α-amylase activity. Formononetin showed the largest activity. Compared with LC–MS/MS, MALDI-TOF-MS is a quick and convenient method; results can be obtained within minutes; and only minor sample amounts are required which allows us to analyse mixtures of substances without preliminary separation. However, the inability to distinguish between isomers is the main limitation of the method. KW - Enzyme KW - MALDI-TOF-MS KW - LC-MS/MS KW - Massenspektrometrie PY - 2023 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-577128 DO - https://doi.org/https://doi.org/10.1111/ijfs.16491 SN - 0950-5423 VL - 58 IS - 7 SP - 3902 EP - 3911 PB - Wiley & Sons CY - Hoboken, NJ, USA AN - OPUS4-57712 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - THES A1 - Ecke, Alexander T1 - Integrated Diagnostics of Pharmaceutical Contaminants in Water Supply and Management Systems N2 - The contamination of drinking water with pharmaceuticals represents a severe health risk. In order to monitor the drinking water quality continuously and enable quick countermeasures in case of contamination, novel sensors are required. Here, immunoanalytical methods based on the binding of the analyte to highly selective antibodies can be helpful. In this work, magnetic bead-based immunoassays (MBBAs) have been developed for the detection of two relevant contaminants of drinking water: diclofenac (DCF) and amoxicillin (AMX). In case of the latter, not only the parent drug is of interest in the risk assessment but also its hydrolysis products (HPs). In a comprehensive study, the influence of external factors and intrinsic properties of the water on the rate of hydrolysis was investigated. As the hydrolysis of AMX further impacts the recognition by the antibody, a strategy to analyze samples with unknown hydrolysis degree of AMX was established employing the enzyme β-lactamase in sample preparation. For both analytes, the MBBAs enable the fast quantification with results obtained in less than one hour which represents a major improvement over conventional immunoassays like the enzyme-linked immunosorbent assay (ELISA). Compared to the respective ELISAs with the same antibodies, the MBBAs further exhibit improved analytical parameters such as a broader measurement range and lower limits of detection. Due to the magnetic properties of the beads that serve as a platform for the assays, they are suitable for the mobile and automated detection at the point-of-care. An integrated diagnostic system was designed in which electrochemical detection with chronoamperometry on a microfluidic chip allows for further miniaturization of the system to enable monitoring of the drinking water quality online in water supply pipes at waterworks. N2 - Die Kontamination von Trinkwasser mit Arzneimitteln stellt eine ernste Gesundheitsgefahr dar. Um die Trinkwasserqualität kontinuierlich überwachen und im Falle einer Verunreinigung zeitnah reagieren zu können, sind neuartige Sensoren erforderlich. Hier können immunanalytische Methoden, die auf der Bindung des Analyten an hochselektive Antikörper beruhen, hilfreich sein. In dieser Arbeit wurden magnetpartikelbasierte Immunoassays (MBBAs) für zwei relevante Kontaminanten des Trinkwassers entwickelt: Diclofenac (DCF) und Amoxicillin (AMX). Bei letzterem erwiesen sich neben der Ausgangsverbindung auch dessen Hydrolyseprodukte (HPs) als relevant für die Gefährdungsbeurteilung. In einer umfassenden Studie wurde der Einfluss von externen Faktoren und intrinsischen Eigenschaften des Wassers auf die Hydrolysegeschwindigkeit untersucht. Da die Hydrolyse von AMX auch die Erkennung durch den Antikörper beeinflusst, wurde eine Strategie zur Analyse von Proben mit unbekanntem Hydrolysegrad von AMX unter Verwendung des Enzyms β-Lactamase in der Probenvorbereitung entwickelt. Für beide Analyten ermöglichen die MBBAs eine schnelle Quantifizierung mit Ergebnissen in weniger als einer Stunde, was eine wesentliche Verbesserung gegenüber herkömmlichen Immunoassays wie dem Enzyme-linked Immunosorbent Assay (ELISA) darstellt. Im Vergleich zu den entsprechenden ELISAs mit denselben Antikörpern weisen die MBBAs zudem verbesserte analytische Parameter auf, wie einen breiteren Messbereich und niedrigere Nachweisgrenzen. Aufgrund der magnetischen Eigenschaften der Partikel, die als Plattform für die Assays dienen, eignen sie sich für den mobilen und automatisierten Einsatz vor Ort. Ein integriertes Diagnosesystem, bei dem die elektrochemische Detektion mittels Chronoamperometrie auf einem mikrofluidischen Chip eine weitere Miniaturisierung des Systems ermöglicht, wurde entworfen, um die Überwachung der Trinkwasserqualität online in Wasserwerken zu ermöglichen. KW - Antibiotics KW - Immunoassay KW - Amoxicillin KW - Diclofenac KW - Biosensor KW - Antibodies PY - 2023 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-591901 DO - https://doi.org/10.18452/25853 SP - 1 EP - 129 CY - Berlin AN - OPUS4-59190 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Tscheuschner, Georg A1 - Ponader, Marco A1 - Raab, Christopher A1 - Weider, Prisca S. A1 - Hartfiel, Reni A1 - Kaufmann, Jan Ole A1 - Völzke, Jule L. A1 - Bosc-Bierne, Gaby A1 - Prinz, Carsten A1 - Schwaar, T. A1 - Andrle, Paul A1 - Bäßler, Henriette A1 - Nguyen, Khoa A1 - Zhu, Y. A1 - Mey, A. S. J. S. A1 - Mostafa, A. A1 - Bald, I. A1 - Weller, Michael G. T1 - Efficient Purification of Cowpea Chlorotic Mottle Virus by a Novel Peptide Aptamer N2 - The cowpea chlorotic mottle virus (CCMV) is a plant virus explored as a nanotechnological platform. The robust self-assembly mechanism of its capsid protein allows for drug encapsulation and targeted delivery. Additionally, the capsid nanoparticle can be used as a programmable platform to display different molecular moieties. In view of future applications, efficient production and purification of plant viruses are key steps. In established protocols, the need for ultracentrifugation is a significant limitation due to cost, difficult scalability, and safety issues. In addition, the purity of the final virus isolate often remains unclear. Here, an advanced protocol for the purification of the CCMV from infected plant tissue was developed, focusing on efficiency, economy, and final purity. The protocol involves precipitation with PEG 8000, followed by affinity extraction using a novel peptide aptamer. The efficiency of the protocol was validated using size exclusion chromatography, MALDI-TOF mass spectrometry, reversed-phase HPLC, and sandwich immunoassay. Furthermore, it was demonstrated that the final eluate of the affinity column is of exceptional purity (98.4%) determined by HPLC and detection at 220 nm. The scale-up of our proposed method seems to be straightforward, which opens the way to the large-scale production of such nanomaterials. This highly improved protocol may facilitate the use and implementation of plant viruses as nanotechnological platforms for in vitro and in vivo applications. N2 - Das Cowpea Chlorotic Mottle Virus (CCMV) ist ein Pflanzenvirus, das als nanotechnologische Plattform erforscht wird. Der robuste Selbstorganisationsmechanismus seines Kapsidproteins ermöglicht die Verkapselung und gezielte Abgabe von Medikamenten. Darüber hinaus kann das Kapsid-Nanopartikel als programmierbare Plattform für die Präsentation verschiedener molekularer Komponenten verwendet werden. Im Hinblick auf künftige Anwendungen ist eine effiziente Produktion und Reinigung von Pflanzenviren von entscheidender Bedeutung. In etablierten Protokollen stellt die notwendige Ultrazentrifugation aufgrund von Kosten, schwieriger Skalierbarkeit und Sicherheitsaspekten eine erhebliche Einschränkung dar. Darüber hinaus bleibt die Reinheit des endgültigen Virusisolats oft unklar. Hier wurde ein fortschrittliches Protokoll für die Reinigung von CCMV aus infiziertem Pflanzengewebe entwickelt, wobei der Schwerpunkt auf Effizienz, Wirtschaftlichkeit und Reinheit lag. Das Protokoll beinhaltet eine Fällung mit Polyethylenglycol (PEG 8000), gefolgt von einer Affinitätsextraktion mit einem neuartigen Peptid-Aptamer. Die Effizienz des Protokolls wurde mithilfe von Größenausschluss-Chromatographie (SEC), MALDI-TOF-Massenspektrometrie, Umkehrphasen-HPLC und Sandwich-Immunoassay validiert. Darüber hinaus wurde nachgewiesen, dass das endgültige Eluat der Affinitätssäule eine außergewöhnliche Reinheit (98,4 %) aufweist, die durch HPLC und Detektion bei 220 nm bestimmt wurde. Die Skalierung der von uns vorgeschlagenen Methode scheint einfach zu sein, was den Weg für eine größer angelegte Produktion solcher Nanomaterialien ebnet. Dieses stark verbesserte Protokoll könnte die Verwendung und Umsetzung von Pflanzenviren als nanotechnologische Plattformen für In-vitro- und In-vivo-Anwendungen erleichtern. KW - Affinity chromatography KW - Nanoparticles KW - Nanoscience KW - Carrier protein KW - Encapsulation KW - Combinatorial peptide library KW - Peptide binder KW - Vigna unguiculata KW - Augenbohne KW - Schlangenbohne KW - Pflanzenvirus KW - Plant virus KW - Upscaling KW - Commercialization KW - Reference material KW - Nanocarrier PY - 2023 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-572645 DO - https://doi.org/10.3390/v15030697 VL - 15 IS - 3 SP - 1 EP - 24 PB - MDPI CY - Basel, Schweiz AN - OPUS4-57264 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -