Short Peptide and Amino Acid-Based Supramolecular Ionogels and Eutectogels.

The capability of peptide and amino acid-based molecules to act as ionogelators and eutectogelators entrapping ionic liquids (ILs) and deep eutectic solvents (DESs) forming ionogels and eutectogels has gathered attention in recent decades. The self-assembly process, primarily driven by non-covalent interactions as hydrogen bonding, remains serendipitous in nature. This review provides a comprehensive and detailed report on self-assembly of unmodified and modified amino acids and peptides in the non-conventional solvents, ILs and DESs. Understanding these processes holds great promise for the development of innovative soft-materials, and to the progress of supramolecular systems in non-conventional solvent environments.

Effect of Polymer Hydrophobicity in the Performance of Hybrid Gel Gas Sensors for E-Noses.

Relative humidity (RH) is a common interferent in chemical gas sensors, influencing their baselines and sensitivity, which can limit the performance of e-nose systems. Tuning the composition of the sensing materials is a possible strategy to control the impact of RH in gas sensors. Hybrid gel materials used as gas sensors contain self-assembled droplets of ionic liquid and liquid crystal molecules encapsulated in a polymeric matrix. In this work, we assessed the effect of the matrix hydrophobic properties in the performance of hybrid gel materials for VOC sensing in humid conditions (50% RH). We used two different polymers, the hydrophobic PDMS and the hydrophilic bovine gelatin, as polymeric matrices in hybrid gel materials containing imidazolium-based ionic liquids, [BMIM][Cl] and [BMIM][DCA], and the thermotropic liquid crystal 5CB. Better accuracy of VOC prediction is obtained for the hybrid gels composed of a PDMS matrix combined with the [BMIM][Cl] ionic liquid, and the use of this hydrophobic matrix reduces the effect of humidity on the sensing performance when compared to the gelatin counterpart. VOCs interact with all the moieties of the hybrid gel multicomponent system; thus, VOC correct classification depends not only on the polymeric matrix used, but also on the IL selected, which seems to be key to achieve VOCs discrimination at 50% RH. Thus, hybrid gels' tunable formulation offers the potential for designing complementary sensors for e-nose systems operable under different RH conditions.

Native, engineered and de novo designed ligands targeting the SARS-CoV-2 spike protein.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the deadly coronavirus disease 2019 (Covid-19) and is a concerning hazard to public health. This virus infects cells by establishing a contact between its spike protein (S-protein) and host human angiotensin-converting enzyme 2 (hACE2) receptor, subsequently initiating viral fusion. The inhibition of the interaction between the S-protein and hACE2 has immediately drawn attention amongst the scientific community, and the S-protein was considered the prime target to design vaccines and to develop affinity ligands for diagnostics and therapy. Several S-protein binders have been reported at a fast pace, ranging from antibodies isolated from immunised patients to de novo designed ligands, with some binders already yielding promising in vivo results in protecting against SARS-CoV-2. Natural, engineered and designed affinity ligands targeting the S-protein are herein summarised, focusing on molecular recognition aspects, whilst identifying preferred hot spots for ligand binding. This review serves as inspiration for the improvement of already existing ligands or for the design of new affinity ligands towards SARS-CoV-2 proteins. Lessons learnt from the Covid-19 pandemic are also important to consolidate tools and processes in protein engineering to enable the fast discovery, production and delivery of diagnostic, prophylactic, and therapeutic solutions in future pandemics.

Nanoscale Events on Cyanobiphenyl-Based Self-Assembled Droplets Triggered by Gas Analytes.

Liquid crystals (LCs) are prime examples of dynamic supramolecular soft materials. Their autonomous self-assembly at the nanoscale level and the further nanoscale events that give rise to unique stimuli-responsive properties have been exploited for sensing purposes. One of the key features to employ LCs as sensing materials derives from the fine-tuning between stability and dynamics. This challenging task was addressed in this work by studying the effect of the alkyl chain length of cyanobiphenyl LCs on the molecular self-assembled compartments organized in the presence of ionic liquid molecules and gelatin. The resulting multicompartment nematic and smectic gels were further used as volatile organic compound chemical sensors. The LC structures undergo a dynamic sequence of phase transitions, depending on the nature of the LC component, yielding a variety of optical signals, which serve as optical fingerprints. In particular, the materials incorporating smectic compartments resulted in unexpected and rich optical textures that have not been reported previously. Their sensing capability was tested in an in-house-assembled electronic nose and further assessed via signal collection and machine-learning algorithms based on support vector machines, which classified 12 different gas analytes with high accuracy scores. Our work expands the knowledge on controlling LC self-assembly to yield fast and autonomous accurate chemical-sensing systems based on the combination of complex nanoscale sensing events with artificial intelligence tools.

Affinity Tags in Protein Purification and Peptide Enrichment: An Overview.

The reversible interaction between an affinity ligand and a complementary receptor has been widely explored in purification systems for several biomolecules. The development of tailored affinity ligands highly specific toward particular target biomolecules is one of the options in affinity purification systems. However, both genetic and chemical modifications in proteins and peptides widen the application of affinity ligand-tag receptors pairs toward universal capture and purification strategies. In particular, this chapter will focus on two case studies highly relevant for biotechnology and biomedical areas, namely the affinity tags and receptors employed on the production of recombinant fusion proteins, and the chemical modification of phosphate groups on proteins and peptides and the subsequent specific capture and enrichment, a mandatory step before further proteomic analysis.

Versatile and Tunable Poly(Ethylene Glycol)-Based Hydrogels Crosslinked through the Ugi Reaction.

The four-component Ugi condensation reaction has been investigated to assemble chemically crosslinked hydrogels using multivalent star-shaped poly(ethylene glycol) components. The resulting biocompatible hydrogels are highly versatile in composition and function. It is shown that acid, aldehyde, and cyanide components can be varied yielding materials with precise structure and tunable stiffness. Additionally, the resulting hydrogels were proven extremely robust to consecutive drying-swelling cycles. This property was explored to develop a reversible humidity colorimetric sensor gel. Overall, this work demonstrates the application of the four-component Ugi reaction as a powerful tool to quickly generate crosslinked gels with precise control in chemical composition.

Natural Multimerization Rules the Performance of Affinity-Based Physical Hydrogels for Stem Cell Encapsulation and Differentiation.

Tissue engineering and stem cell research greatly benefit from cell encapsulation within hydrogels as it promotes cell expansion and differentiation. Affinity-triggered hydrogels, an appealing solution for mild cell encapsulation, rely on selective interactions between the ligand and target and also on the multivalent presentation of these two components. Although these hydrogels represent a versatile option to generate dynamic, tunable, and highly functional materials, the design of hydrogel properties based on affinity and multivalency remains challenging and unstudied. Here, the avidin-biotin affinity pair, with the highest reported affinity constant, is used to address this challenge. It is demonstrated that the binding between the affinity hydrogel components is influenced by the multivalent display selected. In addition, the natural multivalency of the interaction must be obeyed to yield robust multicomponent synthetic protein hydrogels. The hydrogel's resistance to erosion depends on the right stoichiometric match between the hydrogel components. The developed affinity-triggered hydrogels are biocompatible and support encapsulation of induced pluripotent stem cells and their successful differentiation into a neural cell line. This principle can be generalized to other affinity pairs using multimeric proteins, yielding biomaterials with controlled performance.

Magnetic Precipitation: A New Platform for Protein Purification.

One of the trends in downstream processing comprises the use of "anything-but-chromatography" methods to overcome the current downfalls of standard packed-bed chromatography. Precipitation and magnetic separation are two techniques already proven to accomplish protein purification from complex media, yet never used in synergy. With the aim to capture antibodies directly from crude extracts, a new approach combining precipitation and magnetic separation is developed and named as affinity magnetic precipitation. A precipitation screening, based on the Hofmeister series, and a commercial precipitation kit are tested with affinity magnetic particles to assess the best condition for antibody capture from human serum plasma and clarified cell supernatant. The best conditions are obtained when using PEG3350 as precipitant at 4 °C for 1 h, reaching 80% purity and 50% recovery of polyclonal antibodies from plasma, and 99% purity with 97% recovery yield of anti-TNFα mAb from cell supernatants. These results show that the synergetic use of precipitation and magnetic separation can represent an alternative for the efficient capture of antibodies.

Phosphopeptide Enrichment Using Various Magnetic Nanocomposites: An Overview.

Magnetic nanocomposites are hybrid structures consisting of an iron oxide (Fe3O4/γ-Fe2O3) superparamagnetic core and a coating shell which presents affinity for a specific target molecule. Within the scope of phosphopeptide enrichment, the magnetic core is usually first functionalized with an intermediate layer of silica or carbon to improve dispersibility and increase specific area, and then with an outer layer of a phosphate-affinity material. Fe3O4-coating materials include metal oxides, rare earth metal-based compounds, immobilized-metal ions, polymers, and many others. This chapter provides a generic overview of the different materials that can be found in literature and their advantages and drawbacks.

Affinity tags in protein purification and peptide enrichment: an overview.

The reversible interaction between an affinity ligand and a complementary receptor has been widely explored in purification systems for several biomolecules. The development of tailored affinity ligands highly specific towards particular target biomolecules is one of the options in affinity purification systems. However, both genetic and chemical modifications on proteins and peptides widen the application of affinity ligand-tag receptor pairs towards universal capture and purification strategies. In particular, this chapter will focus on two case studies highly relevant for biotechnology and biomedical areas, namely, the affinity tags and receptors employed on the production of recombinant fusion proteins and the chemical modification of phosphate groups on proteins and peptides and the subsequent specific capture and enrichment, a mandatory step before further proteomic analysis.

The interaction of polymer-coated magnetic nanoparticles with seawater.

Laboratory studies were conducted to evaluate the interaction between bare and polymer-coated magnetic nanoparticles (MNPs) with various environmentally relevant carrying solutions including natural oceanic seawater with and without addition of algal exopolymeric substances (EPS). The MNPs were coated with three different stabilising agents, namely gum Arabic (GA-MNP), dextran (D-MNP) and carboxymethyl-dextran (CMD-MNP). The colloidal stability of the suspensions was evaluated over 48 h and we demonstrated that: (i) hydrodynamic diameters increased over time regardless of carrying solution for all MNPs except the GA-coated ones; however, the relative changes were carrying solution- and coat-dependent; (ii) polydispersity indexes of the freshly suspended MNPs are below 0.5 for all coated MNPs, unlike the much higher values obtained for the uncoated MNPs; (iii) freshly prepared MNP suspensions (both coated and uncoated) in Milli-Q (MQ) water show high colloidal stability as indicated by zeta-potential values below -30 mV, which however decrease in absolute value within 48 h for all MNPs regardless of carrying solution; (iv) EPS seems to "stabilise" the GA-coated and the CMD-coated MNPs, but not the uncoated or the D-coated MNPs, which form larger aggregates within 48 h; (v) despite this aggregation, iron (Fe)-leaching from MNPs is sustained over 48h, but remained within the range of 3-9% of the total iron-content of the initially added MNPs regardless of suspension media and capping agent. The environmental implications of our findings and biotechnological applicability of MNPs are discussed.

Challenges and opportunities in the purification of recombinant tagged proteins.

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development.

In situ magnetic separation of antibody fragments from Escherichia coli in complex media.

BACKGROUND: In situ magnetic separation (ISMS) has emerged as a powerful tool to overcome process constraints such as product degradation or inhibition of target production. In the present work, an integrated ISMS process was established for the production of his-tagged single chain fragment variable (scFv) D1.3 antibodies ("D1.3") produced by E. coli in complex media. This study investigates the impact of ISMS on the overall product yield as well as its biocompatibility with the bioprocess when metal-chelate and triazine-functionalized magnetic beads were used. RESULTS: Both particle systems are well suited for separation of D1.3 during cultivation. While the triazine beads did not negatively impact the bioprocess, the application of metal-chelate particles caused leakage of divalent copper ions in the medium. After the ISMS step, elevated copper concentrations above 120 mg/L in the medium negatively influenced D1.3 production. Due to the stable nature of the model protein scFv D1.3 in the biosuspension, the application of ISMS could not increase the overall D1.3 yield as was shown by simulation and experiments. CONCLUSIONS: We could demonstrate that triazine-functionalized beads are a suitable low-cost alternative to selectively adsorb D1.3 fragments, and measured maximum loads of 0.08 g D1.3 per g of beads. Although copper-loaded metal-chelate beads did adsorb his-tagged D1.3 well during cultivation, this particle system must be optimized by minimizing metal leakage from the beads in order to avoid negative inhibitory effects on growth of the microorganisms and target production. Hereby, other types of metal chelate complexes should be tested to demonstrate biocompatibility. Such optimized particle systems can be regarded as ISMS platform technology, especially for the production of antibodies and their fragments with low stability in the medium. The proposed model can be applied to design future ISMS experiments in order to maximize the overall product yield while the amount of particles being used is minimized as well as the number of required ISMS steps.

In vitro studies with mammalian cell lines and gum arabic-coated magnetic nanoparticles.

Iron oxide magnetic nanoparticles (MNPs) were synthesized by the chemical co-precipitation method and coated with gum arabic (GA) by physical adsorption and covalent attachment. Cultures of mammalian cell lines (HEK293, CHO and TE671) were grown in the presence of uncoated and GA-coated MNPs. Cellular growth was followed by optical microscopy in order to assess the proportion of cells with particles, alterations in cellular density and the presence of debris. The in vitro assays demonstrated that cells from different origins are affected differently by the presence of the nanoparticles. Also, the methods followed for GA coating of MNPs endow distinct surface characteristics that probably underlie the observed differences when in contact with the cells. In general, the nanoparticles to which the GA was adsorbed had a smaller ability to attach to the cells' surface and to compromise the viability of the cultures.

An historical overview of drug discovery.

Drug Discovery in modern times straddles three main periods. The first notable period can be traced to the nineteenth century where the basis of drug discovery relied on the serendipity of the medicinal chemists. The second period commenced around the early twentieth century when new drug structures were found, which contributed for a new era of antibiotics discovery. Based on these known structures, and with the development of powerful new techniques such as molecular modelling, combinatorial chemistry, and automated high-throughput screening, rapid advances occurred in drug discovery towards the end of the century. The period also was revolutionized by the emergence of recombinant DNA technology, where it became possible to develop potential drugs target candidates. With all the expansion of new technologies and the onset of the "Omics" revolution in the twenty-first century, the third period has kick-started with an increase in biopharmaceutical drugs approved by FDA/EMEA for therapeutic use.

Affinity chromatography: history, perspectives, limitations and prospects.

Biomolecule separation and purification has until very recently steadfastly remained one of the more empirical aspects of modem biotechnology. Affinity chromatography, one of several types of adsorption chromatography, is particularly suited for the efficient isolation of biomolecules. This technique relies on the adsorbent bed material that has biological affinity for the substance to be isolated. This review is intended to place affinity chromatography in historical perspective and describe the current status, limitations and future prospects for the technique in modern biotechnology.

Rationally designed ligands for use in affinity chromatography: an artificial protein L.

Synthetic affinity ligands can circumvent the drawbacks of natural immunoglobulin (Ig)-binding proteins by imparting resistance to chemical and biochemical degradation and to in situ sterilization, as well as ease and low cost of production. Protein L (PpL), isolated from Peptostreptococcus magnus strains, interacts with the Fab (antigen-binding fragment) portion of Igs, specifically with kappa light chains, and represents an almost universal ligand for the purification of antibodies. The concepts of rational design and solid-phase combinatorial chemistry were used for the discovery of a synthetic PpL mimic affinity ligand. The procedure presented in this chapter represents a general approach with the potential to be applied to different systems and target proteins.

Design, synthesis, and screening of biomimetic ligands for affinity chromatography.

Affinity chromatography is ideally suited to the purification of pharmaceutical proteins due to its unique bio-specificity characteristics. Tailor-made affinity ligands that represent a promising class of synthetic affinity ligands have been developed to target specific proteins and designed to mimic peptidal templates, natural biological recognition motifs, or complementary surface-exposed residues. These biomimetic ligands have been generated by a combination of rational design, combinatorial library synthesis, and subsequent screening of potential leads against target proteins. Small ligands based on a triazine scaffold also present exceptional selectivity and stability, which allows their use in harsh manufacturing environments.