Affinity chromatography is one of the most common techniques employed at the industrial-scale for antibody purification. In particular, the purification of human immunoglobulin G (hIgG) has gained relevance with the immobilization of its natural binding counterpart—Staphylococcus aureus Protein A (SpA) or with the recent development of biomimetic affinity ligands, namely triazine-based ligands. These ligands have been developed in order to overcome economic and leaching issues associated to SpA. The most recent triazine-based ligand—TPN-BM, came up as an analogue of 2-(3-amino-phenol)-6-(4-amino-1-naphthol)-4-chloro-sym-triazine ligand also known as ligand 22/8 with improved physico-chemical properties and a greener synthetic route. This work intends to evaluate the potential of TPN-BM as an alternative affinity ligand towards antibody recognition and binding, namely IgG, at an atomic level, since it has already been tested, after immobilization onto chitosan-based monoliths and demonstrated interesting affinity behaviour for this purpose. Herein, combining automated molecular docking and molecular dynamics simulations it was predicted that TPN-BM has high propensity to bind IgG through the same binding site found in the crystallographic structure of SpA_IgG complex, as well as theoretically predicted for ligand 22/8_IgG complex. Furthermore, it was found that TPN-BM established preferential interactions with aromatic residues at the Fab domain (Trp 50, Tyr 53, Tyr 98 and Trp 100), while in the Fc domain the main interactions are based on hydrogen bonds with pH sensitive residues at operational regime for binding and elution like histidines (His 460, His 464, His 466). Moreover, the pH dependence of TPN-BM_IgG complex formation was more evident for the Fc domain, where at pH 3 the protonation state and consequently the charge alteration of histidine residues located at the IgG binding site induced ligand detachment which explains the optimal elution condition at this pH observed experimentally.

A novel affinity “tag–receptor” pair was developed as a generic platform for the purification of fusion proteins. The hexapeptide RKRKRK was selected as the affinity tag and fused to green fluorescent protein (GFP). The DNA fragments were designed, cloned in Pet-21c expression vector and expressed in E. coli host as soluble protein. A solid-phase combinatorial library based on the Ugi reaction was synthesized: 64 affinity ligands displaying complementary functionalities towards the designed tag. The library was screened by affinity chromatography in a 96-well format for binding to the RKRKRK-tagged GFP protein. Lead ligand A7C1 was selected for the purification of RKRKRK fusion proteins. The affinity pair RKRKRK-tagged GFP with A7C1 emerged as a promising solution (Ka of 2.45×105 M−1). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.

The green fluorescent protein (GFP) is widely employed to report on a variety of molecular phenomena, but its selective recovery is hampered by the lack of a low-cost and robust purification alternative. This work reports an integrated approach combining rational design and experimental validation toward the optimization of a small fully-synthetic ligand for GFP purification. A total of 56 affinity ligands based on a first-generation lead structure were rationally designed through molecular modeling protocols. The library of ligands was further synthesized by solid-phase combinatorial methods based on the Ugi reaction and screened against Escherichia coli extracts containing GFP. Ligands A4C2, A5C5 and A5C6 emerged as the new lead structures based on the high estimated theoretical affinity constants and the high GFP binding percentages and enrichment factors. The elution of GFP from these adsorbents was further characterized, where the best compromise between mild elution conditions, yield and purity was found for ligands A5C5 and A5C6. These were tested for purifying a model GFP-fusion protein, where ligand A5C5 yielded higher protein recovery and purity. The molecular interactions between the lead ligands and GFP were further assessed by molecular dynamics simulations, showing a wide range of potential hydrophobic and hydrogen-bond interactions.

Improved thermoelectric properties of Aluminum Zinc Oxide (AZO) thin films deposited by radio frequency (RF) and pulsed Direct Current (DC) magnetron sputtering at room temperature are reported. In both techniques films were deposited using sintered and non-sintered targets produced from nano-powders. It is confirmed that both the Al doping concentration and film thickness control the thermoelectric, optical and structural properties of these films. Seebeck coefficients up to −134 μV K−1 and electrical conductivities up to 4 × 104 (Ω m)−1 lead to power factors up to 4 × 10−4 W mK−2, which is above the state-of-the-art for similar materials, almost by a factor of three. The thermoelectric I–V response of an optimized AZO element with a planar geometry was measured and a maximum power output of 2.3 nW, for a temperature gradient of 20 K near room temperature, was obtained. Moreover, the low thermal conductivity (<1.19 W mK−1) yields a ZT value above 0.1. This is an important result as it is at least three times higher than the ZT found in the literature for AZO, at room temperature, opening new doors for applications of this inexpensive, abundant and environmental friendly material, in a new era of thermoelectric devices.

Arabinanase is a glycosyl hydrolase that is able to cleave the glycosidic bonds of α-1,5-L-arabinan, releasing arabino-oligosaccharides and L-arabinose. The enzyme has two domains, an N-terminal catalytic domain with a characteristic β-propeller fold and a C-terminal domain whose function is unknown. A calcium ion, located near the catalytic site, serves to stabilize the N-terminal domain, but it has also been proposed to play a key role in the enzyme mechanism. The present work describes the structure of an inactive mutant of the wild-type enzyme (H318Q) and in which the calcium ion has been adventitiously replaced by nickel. These structural studies, together with functional and modelling studies, clearly support the role of the calcium ion in the overall reaction mechanism.

Pulsed field gradient spin echo high resolution magic angle spinning nuclear magnetic resonance spectroscopy is a powerful technique to characterize confined biosystems. We used this approach to assess the diffusion of solvent and reaction species within sol–gel matrices differing in enzyme loading.

Spatial and temporal control of molecular mechanisms can be achieved using photolabile bonds that connect biomolecules to protective caging groups, which can be cleaved upon irradiation of a specific wavelength, releasing the biomolecule ready-to-use. Here we apply and improve a previously reported strategy to tightly control in vitro transcription reactions. The strategy involves two caging molecules that block both ATP and GTP nucleotides. Additionally, we designed a molecular beacon complementary to the synthesized mRNA to infer its presence through a light signal. Upon release of both nucleotides through a specific monochromatic light (390 and 325 nm) we attain a light signal indicative of a successful in vitro transcription reaction. Similarly, in the absence of irradiation, no intense fluorescence signal was obtained. We believe this strategy could further be applied to DNA synthesis or the development of logic gates.

Cancer development is amultistep process in which exosomes play important roles. Exosomes are small vesicles formed in vesicular bodies in the endosomal network. The major role of exosomes seems to be the transport of bioactive molecules between cells. Depending on the cell of origin, exosomes are implicated in the regulation of several cellular events, with phenotypic consequences in recipient cells. Cancer derived exosomes (CCEs) are important players in the formation of the tumour microenvironment by (i) enabling the escape of tumour cells to immunological system and help initiating the inflammatory response; (ii) acting in the differentiation of fibroblasts and mesenchymal cells into myofibroblasts; (iii) triggering the angiogenic process; and (iv) enhancing the metastatic evolution of the tumour by promoting epithelial to mesenchymal transformation of tumour cells and by preparing the tumour niche in the new anatomical location. Since the finding that exosomes content resembles that of the cell of origin, they may be regarded as suitable biomarkers for cancer diagnosis, allowing for diagnosis and prognosis via a minimal invasive procedure. Exosome involvement in cancer may open new avenues regarding therapeutics, such as vectors for targeted drug delivery.

This chapter focuses on several sensing strategies and major recent advances in the use of gold nanoparticles in (bio)sensing of chemical and biological analytes. A brief introduction is presented on relevant properties of gold nanoparticles for sensing, the main types of (bio)chemical sensors, and the main detection techniques, followed by subsections according to sensing methodologies. These include colorimetric sensing and the biobarcode assay, fluorometric-based methods, electric and electrochemical sensing, and, last, more recent and advanced methodologies such as surface plasmon resonance and Raman-based sensors. In closing, relevance is given to advanced methods, featuring extremely high sensitivity and selectivity, down to single-molecule detection. Anisotropic gold nanoparticles have a special role in future developments.