Human serum albumin (HSA) in an important therapeutic agent and disease biomarker, with an increasing market demand. By proteins and drugs that bind to HSA as inspiration, a combinatorial library of 64 triazine-based ligands was rationally designed and screened for HSA binding at physiological conditions. Two triazine-based lead ligands (A3A2 and A6A5), presenting more than 50% HSA bound and high enrichment factors, were selected for further studies. Binding and elution conditions for HSA purification from human plasma were optimized for both ligands. The A6A5 adsorbent yielded a purified HSA sample with 98% purity at 100% recovery yield under mild binding and elution conditions.

Currently most economical and technological bottlenecks in protein production are placed in the down-stream processes. With the aim of increasing the efficiency and reducing the associated costs, variousaffinity ligands have been developed. Affitins are small, yet robust and easy to produce, proteins derivedfrom the archaeal extremophilic “7 kDa DNA-binding” protein family. By means of combinatorial pro-tein engineering and ribosome display selection techniques, Affitins have shown to bind a diversity oftargets. In this work, two previously developed Affitins (anti-lysozyme and anti-IgG) were immobilizedonto magnetic particles to assess their potential for protein purification by magnetic fishing. The opti-mal lysozyme and human IgG binding conditions yielded 58 mg lysozyme/g support and 165 mg IgG/gsupport, respectively. The recovery of proteins was possible in high yield (≥95{%}) and with high purity,namely ≥95{%} and 81{%}, when recovering lysozyme from Escherichia coli supernatant and IgG from humanplasma, respectively. Static binding studies indicated affinity constants of 5.0 × 104M−1and 9.3 × 105M−1for the anti-lysozyme and anti-IgG magnetic supports. This work demonstrated that Affitins, which canbe virtually evolved for any protein of interest, can be coupled onto magnetic particles creating novelaffinity adsorbents for purification by magnetic fishing.

Oleic acid coated iron oxide nanoparticles synthesized by thermal decomposition in organic medium are highly monodisperse but at the same time are unsuitable for biological applications. Ligand-exchange reactions are useful to make their surface hydrophilic. However, these could alter some structural and magnetic properties of the modified particles. Here we present a comprehensive study and comparison of the effects of employing either citric acid (CA) or meso-2,3-dimercaptosuccinic acid (DMSA) ligand-exchange protocols for phase transfer of monodisperse hydrophobic iron oxide nanoparticles produced by thermal decomposition of Fe(acac)3 in benzyl ether. We show the excellent hydrodynamic size distribution and colloidal stability of the hydrophilic particles obtained by the two protocols and confirm that there is a certain degree of oxidation caused by the ligand-exchange. CA revealed to be more aggressive towards the iron oxide surface than DMSA and greatly reduced the saturation magnetization values and initial susceptibility of the resulting particles compared to the native ones. Besides being milder and more straightforward to perform, the DMSA ligand exchange protocol produces MNP chemically more versatile for further functionalization possibilities. This versatility is shown through the covalent linkage of gum Arabic onto MNP-DMSA using carboxyl and thiol based chemical routes and yielding particles with comparable properties.

Fluorescence microscopy and microspectrofluorometry are important tools in the characterization and identification of proteins, offering a great range of applications in conservation science. Because of their high selectivity and sensitivity, the combination of these techniques can be exploited for improved recognition and quantification of proteinaceous binders in paintings and polychromed works of art. The present article explores an analytical protocol integrating fluorescence microscopy and fluorometry for both identification and mapping of proteinaceous binders (in particular egg and glues) in paint samples. The study has been carried out on historically accurate reconstructions simulating the structure and composition of tempera and oil paints containing these binders. To assess the spatial distribution of specific proteins within the paint layers, cross-sections from the reconstructions were analyzed by fluorescence imaging after staining with an exogenous fluorophore. Reference fluorescence spectra for each layer were acquired by a multichannel spectral analyzer and compared after Gaussian deconvolution. The results obtained demonstrated the effectiveness of the integrated protocol, highlighting the potential for the use of fluorescent staining coupled with microspectrofluorometry as a routine diagnostic tool in conservation science. The current work creates a set of fully characterized reference samples for further comparison with those from actual works of art.