Wissenschaftliche Artikel der BAM
Filtern
Dokumenttyp
Sprache
- Englisch (2)
Schlagworte
- Affinity chromatography (1)
- Antibodies (1)
- Classification (1)
- Corundum (1)
- Dendrimer (1)
- Nanomaterial (1)
- Nonspecific binding (NSB) (1)
- Particle size (1)
- Polyglycerol (1)
- Purification (1)
Organisationseinheit der BAM
Nonporous corundum powder, known as an abrasive material in the industry, was functionalized covalently with protein binders to isolate and enrich specific proteins from complex matrices. The materials based on corundum were characterized by TEM, ESEM, BET, DLS, EDS, and zeta potential measurements. The strong Al-O-P bonds between the corundum surface and amino phosphonic acids were used to introduce functional groups for further conjugations. The common crosslinker glutaraldehyde was compared with a hyperbranched polyglycerol (PG) of around 10 kDa. The latter was oxidized with periodate to generate aldehyde groups that can covalently react with the amines of the surface and the amino groups from the protein via a reductive amination process. The amount of bound protein was quantified via aromatic amino acid analysis (AAAA). This work shows that oxidized polyglycerol can be used as an alternative to glutaraldehyde. With polyglycerol, more of the model protein bovine serum albumin (BSA) could be attached to the surface under the same conditions, and lower non-specific binding (NSB) was observed. As a proof of concept, IgG was extracted with protein A from crude human plasma. The purity of the product was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A binding capacity of 1.8 mg IgG per gram of corundum powder was achieved. The advantages of corundum include the very low price, extremely high physical and chemical stability, pressure resistance, favorable binding kinetics, convenient handling, and flexible application.
The VSSA approach has the important advantage over classifying, imaging and counting techniques that it does not involve dispersion protocols. Further, the BET technique as the basis for VSSA determination it is in widespread use, generates low costs and is specified for many commercial materials. Finally, the same equipment allows for a deeper analysis by full isotherm evaluation.
The present deliverable assesses all NanoDefine powders, supplemented by further real-world materials (in total 26 powders), and quantitatively compares the relationship between the median size (by Electron Microscopy – considered as benchmark for the EC nanomaterial definition) vs. the size derived from VSSA. The VSSA method mitigates the challenges of EM to assess the thickness of platelets, but worked as well on fibbers and particles of irregular shapes. A screening strategy is proposed. If applied to the further data from real-world materials as validation set, this screening does achieve a correct classification, leaving only borderline materials for tier 2 assessment.