Search

In this thesis, we describe the preparation of Janus particles with face-separated compartments. These are anisotropic spherical colloidal particles of the inorganic origin. These compartmentalized particles have unique properties, such as different chemistries designed to specifically target bio applications. Biofunctionalized Janus particles with tailored surface chemistry in general have been gathering interest. The dual nature of the surface chemistry of Janus particles can be exploited to immobilize drugs, cell surface targets, and/or other functional molecules on both sides of the particle surface. There have been several reports and studies based on the preparation of Janus materials including several shapes and materials along with their widespread applications since a few years. The use of micro-sized particles with unique “Janus” character has been widely exploited over the years. However, there are certain limitations pertaining to the use of micro-sized particles when bio applications are concerned. In addition, there is a limited amount of research performed with nano-sized particles exhibiting the “Janus” character and their bio applications. In this thesis, we adapted the current state of the art to synthesize nano-sized Janus particles in bulk-quantities and used these particles to demonstrate bio applications including dual protein functionalization, agglomerate-free bacterial separation from mixtures and attachment to the cell surfaces of eukaryotic cells with minimal uptake. First, we established a model system for the scalable preparation of nanoscale Janus particles with dual protein functionalization with the proteins ferritin and streptavidin. We used 80 nm silica NPs (SiNPs) modified with azidosilane to prepare Pickering emulsions with molten wax as the droplet phase. The azide-functionalized SiNPs on the Pickering emulsion droplets were further subjected to face-selective silanization with biotin-polyethylene glycol (PEG) ethoxy silane. Afterwards, we grafted ferritin on the azide-functionalized side via a click-reaction and the biotin groups were conjugated with streptavidin which was labeled with ultra-small gold NPs. In order to elucidate the advantages and limits of our approach, we performed a detailed characterization of the particles at every process step. The results showed that this method represented a scalable platform for the versatile preparation of protein- nanoscale Janus NPs that can potentially be used with a wide variety of proteins. We further took advantage of the established method where protein-protein functionalization at the nanoscale was demonstrated, to prepare Janus SiNPs for bio application on a prokaryote-based system. We presented a scalable method for designing magnetic Janus NPs which are capable of performing bacterial capture while preventing agglomeration between bacterial cells. To this end, we prepared silica-coated magnetite (Fe3O4) Janus NPs functionalized with a bacteria-specific antibody on one side and PEG chains on the other, using the established wax-in-water emulsion strategy. These magnetic Janus NPs specifically interacted with one type of bacteria from a mixture of bacteria via specific antigen-antibody interactions. Contrary to bacterial capture with isotropically functionalized particles, the bacterial suspensions remained free from cell-NP-cell agglomerates owing to the passivation coating with PEG chains attached to the half of the magnetic NPs pointing away from the bacterial surface after capture. Selective magnetic capture of Escherichia coli (E.coli) cells was achieved from a mixture with Staphylococcus simulans (S.simulans) without compromising bacterial viability and with an efficiency over 80%. This approach is a promising method for rapid and agglomeration-free separation of live bacteria for identification, enrichment and cell counting of bacteria from biological samples. Furthermore, after the successful preparation of Janus NPs for the selective capture of bacteria, we prepared Janus NPs that are designed for the attachment to eukaryotic cell surface molecules with minimal cell uptake. To this end, we synthesized rhodamine-doped SiNPs functionalized with 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) on one hemisphere of the NP surface and high-molecular-weight long-chain PEG on the other one using the wax-Pickering emulsion technique. NP localization was studied with mouse fibroblasts in vitro. In these studies, the Janus NPs were attached to the cell surface and, in contrast to isotropic control particles, only negligible uptake into the cells was observed, even after 24 h of incubation. The study revealed that the prolonged attachment of the Janus NPs is most likely the result of an incomplete macropinocytosis process, and it seems to be independent from caveolae- and receptor-mediated endocytosis. Consequently, by design, these Janus NPs have the potential to firmly anchor onto cell surfaces for extended periods of time which might be utilized in various biotechnological and biomedical applications like cell surface tagging, magnetic manipulation of the cell membrane or non-invasive drug and gene delivery.

Damit eine Symbiose zwischen Ektomykorrhizen und Pflanzen stattfinden kann, muss die Struktur und Funktionalität von Feinwurzeln angepasst werden. Nur so kann der Stoffaustausch von Zucker gegen Nährstoffe und weitere Metabolite stattfinden. Die grundlegenden Prozesse dieser Anpassungen sind dabei in großen Teilen noch unerforscht. Auf dieser Grundlage wurden in vorangegangenen Studien durch genomweite Genexpressionsanalysen drei Pappel Gene identifiziert, deren Transkription in Mykorrhizen 20 – 100 -fach niedriger war. Es handelte sich dabei um ein Kalzium Bindeprotein (Potri.2G2183), einen Aminosäure Transporter (Potri.2G0797) und ein Enzym aus dem Fettstoffwechsel (Potri.9G1040). Um die Proteine zu charakterisieren und den Mechanismus der Regulierung herauszufinden, wurden die Promotoren in dieser Arbeit auf mögliche cis-Elemente untersucht. Dafür wurden Promotor-Reporter Konstrukte mit Fluoreszenzproteinen hergestellt und dann mittels Agrobakterien zur Behandlung von Komposit Pappeln verwendet. Als Grundlage für diese Analyse konnte im Rahmen dieser Arbeit durch einen Wechsel von pPLV zum pCXUN basierten Vektorrückrat die Transformationseffizienz der Pappeln um 69.8% erhöht werden. Weiterhin wurde herausgefunden, dass weder die Orientierung der Fluoreszenz Kassetten auf der T – DNA, noch deren Größe einen messbaren Einfluss auf die Pflanzentransformationseffizienz haben. Dabei konnten Promoter Bereiche von Potri.2G2183 und Potri.2G0797 erfolgreich mittels PCR amplifiziert und trunkiert werden. Zur weiteren Charakterisierung der Promotoren, wurden diese und ihre Trunkierungen in eine eGFP-NLS Expressionskassette kloniert. Um potenzielle cis – Elemente durch Auswertung visueller Daten zu finden, wurde eine konstitutiv exprimierende tdTomato-NLS Expressionskassette in Tandem mit der Promoter Kassette geschaltet (Grün: Rot Verhältnis). Diese Verhältnisse können gegebenenfalls Aufschluss über Änderungen in der Genexpression durch die Mykorrhizierungen geben. Als Alternative zu diesen so genannten Doppelmarker Konstrukten aus eGFP und tdTomato wurde das Timer Protein DsRED-E5 etabliert. Weil das grüne Signal im Verhältnis zum roten Signal im Falle des Timer Proteins sehr schwach war, wurde die Bildaufnahme durch Variierung des der Kameraeinstellungen optimiert. In anschließenden Studien muss diese Methode nun auf Anwendbarkeit in Mykorrhizen getestet werden. Insgesamt muss die visuelle Quantifizierung noch weiter optimiert werden. Auch subzälluläre Lokalisierung der Gene basierend auf transienter Expression in Tabakblättern wurde durchgeführt. Da hier keine eindeutigen Signale detektiert werden konnten, muss das System weiter angepasst und optimiert werden.