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Organisationseinheit der BAM
In dieser Arbeit wurden drei verschiedene Nanomaterialien auf ihre Bindungsfähigkeit zu Proteinen untersucht. Zu Beginn standen dabei die Herstellung stabiler Dispersionen der einzelnen Nanopartikel und die Stabilität der gebildeten Konjugate im Vordergrund. Der Nachweis einer erfolgreichen Konjugatbildung, sprich der Beschichtung von Nanopartikel mit Proteinen, wurde sowohl qualitativ mittels DLS-Messungen als auch über quantitative Protein-Bestimmungen erbracht. Für die Quantifizierung konnten verschiedene Methoden eingesetzt werden. Neben der klassischen Vorgehensweise, welche indirekt über die Quantifizierung von ungebundenem Protein im Überstand erfolgt, konnten ihm Rahmen dieser Arbeit verschiedene direkte Bestimmungsmethoden entwickelt werden. So wurden mittels kolorimetrischer Tests, wie dem BCA-Assay und dem Bradford-Assay, Nanodiamantdispersionen mit Hilfe einer Korrekturwellenlänge vermessen und quantifiziert. Ebenso zum Einsatz kam die Methode der Aminosäureanalytik, welche aufgrund ihrer guten Rückführbarkeit auf Aminosäurestandards Ergebnisse mit hoher Richtigkeit generieren kann und ebenso die Detektion kleiner Proteinmengen möglich macht.
Nach den erfolgten quantitativen Betrachtungen wurden die Protein-beschichteten Nanopartikel auf ihre Anwendbarkeit als Analoga von Virus-like Particles (VLP) bei einer Immunisierung zur Gewinnung von polyklonalen Antikörpern gegen humanes Ceruloplasmin in Kaninchen überprüft. Es konnte mittels ELISA gezeigt werden, dass die Konjugate erfolgreich für die Herstellung von Antikörpern eingesetzt werden können und im zeitlichen Verlauf einer Immunisierung eine Steigerung des Antikörper-Titers zu erreichen ist.
Laser tissue soldering (LTS) based on indocyanine green (ICG)-mediated heat- denaturation of proteins might be a promising alternative technique for micro-suturing, but up to now the Problem of too weak shear strength of the solder welds in comparison to sutures is not solved. Earlier reports gave promising results showing that solder supported by carrier materials can enhance the cohesive strength of the liquid solder. In these studies, the solder was applied to the carriers by dip coating. Higher reliability of the connection between the solder and the carrier material is expected when the solder is bound covalently to the carrier material. In the present study a poly(ether imide) (PEI) membrane served as carrier material and ICG- supplemented albumin as solder substrate. The latter was covalently coupled to the carrier membrane under physiological conditions to prevent structural protein changes. As laser source a diode continuous-wave laser emitting at 808 nm with intensities between 250mW and 1500mW was utilized. The Albumin functionalized carrier membrane was placed onto the tunica media of explanted pig thoracic aortae forming an overlapping area of approximately 0.5×0.5 cm2. All tests were performed in a dry state to prevent laser light absorption by water. Infrared spectroscopy, spectro-photometrical determination of the secondary and Primary amine groups after acid orange II staining, contact angle measurements, and atomic force microscopy proved the successful functionalization of the PEI membrane with albumin. A laser power of 450mW LTS could generate a membrane-blood vessel connection which was characterized by a shear strength of 0.08±0.002MPa, corresponding to 15% of the tensile strength of the native blood vessel. Theoretically, an overlapping zone of 4.1mmaround the entire circumference of the blood vessel could have provided shear strength of the PEI membrane-blood vessel compound identical to the tensile strength of the native blood vessel. These in-vitro results confirmed the beneficial effects of solder reinforcement by carrier membranes, and suggest LTS with covalently bound solders on PEI substrates for further studies in animal models.
The allergenic potential of airborne proteins may be enhanced via post-translational modification induced by air pollutants like ozone (O3) and nitrogen dioxide (NO2). The molecular mechanisms and kinetics of the chemical modifications that enhance the allergenicity of proteins, however, are still not fully understood. Here, protein tyrosine nitration and oligomerization upon simultaneous exposure of O3 and NO2 were studied in coated-wall flow-tube and bulk solution experiments under varying atmospherically relevant conditions (5–200 ppb O3, 5–200 ppb NO2, 45–96% RH), using bovine serum albumin as a model protein. Generally, more tyrosine residues were found to react via the nitration pathway than via the oligomerization pathway. Depending on reaction conditions, oligomer mass fractions and nitration degrees were in the ranges of 2.5–25% and 0.5–7%, respectively. The experimental results were well reproduced by the kinetic multilayer model of aerosol surface and bulk chemistry (KM-SUB). The extent of nitration and oligomerization strongly depends on relative humidity (RH) due to moisture-induced phase transition of proteins, highlighting the importance of cloud processing conditions for accelerated protein chemistry. Dimeric and nitrated species were major products in the liquid phase, while protein oligomerization was observed to a greater extent for the solid and semi-solid phase states of proteins. Our results show that the rate of both processes was sensitive towards ambient ozone concentration but rather insensitive towards different NO2 levels. An increase of tropospheric ozone concentrations in the Anthropocene may thus promote pro-allergic protein modifications and contribute to the observed increase of allergies over the past decades.
Chemical modifications such as nitration and cross-linking may enhance the allergenic potential of proteins. The kinetics and mechanisms of the underlying chemical processes, however, are not yet well understood. Here, we present a size-exclusion chromatography/spectrophotometry method (SEC-HPLC-DAD) that allows a simultaneous detection of mono-, di-, tri-, and higher protein oligomers, as well as their individual nitration degrees (NDs). The ND results of proteins from this new method agree well with the results from an alternative well-established method, for the analysis of tetranitromethane (TNM)- and nitrogen dioxide and ozone (NO2/O3)-nitrated protein samples. Importantly, the NDs for individual oligomer fractions can be obtained from the new method, and also, we provide a proof of principle for the calculation of the concentrations for individual protein oligomer fractions by their determined NDs, which will facilitate the investigation of the kinetics and mechanism for protein tyrosine nitration and cross-linking.
Characterization of silver nanoparticles in cell culture medium containing fetal bovine serum
(2016)
Nanoparticles are being increasingly used in consumer products worldwide, and their toxicological effects are currently being intensely debated. In vitro tests play a significant role in nanoparticle risk assessment, but reliable particle characterization in the cell culture medium with added fetal bovine serum (CCM) used in these tests is not available. As a step toward filling this gap, we report on silver ion release by silver nanoparticles, and changes in the particle radii and in their protein corona when incubated in CCM. Particles of a certified reference material (CRM), p1, and particles of a commercial silver nanoparticle material, p2, were investigated. The colloidal stability of p1 is provided by the surfactants polyethylene glycole-25 glyceryl trioleate and polyethylene glycole-20 sorbitan monolaurate, whereas p2 is stabilized by polyvinylpyrrolidone (PVP). Dialysis of p1 and p2 reveal that their silver ion release rates in CCM are much larger than in water. Particle characterization was performed with asymmetrical flow field-flow fractionation (FFF), small-angle X-ray scattering (SAXS), dynamic light scattering (DLS) and electron microscopy. p1 and p2 have similar hydrodynamic radii of 15 nm and 16 nm, respectively. The silver core radii are 9.2 and 10.2 nm. Gel electrophoresis and subsequent peptide identification reveal that albumin is the main corona component of p1 and p2 after incubation in CCM, which consists of Dulbeccos Modified Eagle Medium with 10% fetal bovine serum added.
Surface-enhanced Raman scattering (SERS) hybrid probes are characterized by the typical spectrum of a reporter molecule. In addition, they deliver information from their biological environment. Here, we report SERS hybrid probes generated by conjugating different reporter molecules to bovine serum albumin (BSA) and using gold nanoparticles as plasmonic core. Advantages of the BSA-conjugate hybrid nanoprobes over other SERS nanoprobes are a high biocompatibility, stabilization of the gold nanoparticles in the biological environment, stable reporter signals, and easy preparation. The coupling efficiencies of the BSA–reporter conjugates were determined by MALDI-TOF-MS. The conjugates' characteristic SERS spectra differ from the spectra of unbound reporter molecules. This is a consequence of the covalent coupling, which leads to altered SERS enhancement and changes in the chemical structures of the reporter and of BSA. The application of the BSA–reporter conjugate hybrid probes in 3T3 cells, including duplex imaging, is demonstrated. Hierarchical cluster analysis and principal components analysis were applied for multivariate imaging using the SERS signatures of the incorporated SERS hybrid nanoprobes along with the spectral information from biomolecules in endosomal structures of cells. The results suggest more successful applications of the SERS hybrid probes in cellular imaging and other unordered high-density bioanalytical sensing.