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.

Impact of the Cationic Moiety of Ionic Liquids on Chemoselective Artificial Olfaction.

Ionogels and derived materials are assemblies of polymers and ionic liquids characterized by high stability and ionic conductivity, making them interesting choices as gas sensors. In this work, we assessed the effect of the ionic liquid moiety to generate ionogels and hybrid gels as electrical and optical gas sensors. Six ionic liquids consisting of a constant anion (chloride) and distinct cationic head groups were used to generate ionogels and hybrid gels and further tested as gas sensors in customized electronic nose devices. In general, ionogel-based sensors yielded higher classification accuracies of standard volatile organic compounds when compared to hybrid material-based sensors. In addition, the high chemical diversity of ionic liquids is further translated to a high functional diversity in analyte molecular recognition and sensing.

Hierarchical self-assembly of a reflectin-derived peptide.

Reflectins are a family of intrinsically disordered proteins involved in cephalopod camouflage, making them an interesting source for bioinspired optical materials. Understanding reflectin assembly into higher-order structures by standard biophysical methods enables the rational design of new materials, but it is difficult due to their low solubility. To address this challenge, we aim to understand the molecular self-assembly mechanism of reflectin's basic unit-the protopeptide sequence YMDMSGYQ-as a means to understand reflectin's assembly phenomena. Protopeptide self-assembly was triggered by different environmental cues, yielding supramolecular hydrogels, and characterized by experimental and theoretical methods. Protopeptide films were also prepared to assess optical properties. Our results support the hypothesis for the protopeptide aggregation model at an atomistic level, led by hydrophilic and hydrophobic interactions mediated by tyrosine residues. Protopeptide-derived films were optically active, presenting diffuse reflectance in the visible region of the light spectrum. Hence, these results contribute to a better understanding of the protopeptide structural assembly, crucial for the design of peptide- and reflectin-based functional materials.

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.

Solvent modulation in peptide sub-microfibers obtained by solution blow spinning.

Peptides possess high chemical diversity at the amino acid sequence level, which further translates into versatile functions. Peptides with self-assembling properties can be processed into diverse formats giving rise to bio-based materials. Peptide-based spun fibers are an interesting format due to high surface-area and versatility, though the field is still in its infancy due to the challenges in applying the synthetic polymer spinning processes to protein fibers to peptides. In this work we show the use of solution blow-spinning to produce peptide fibers. Peptide fiber formation was assisted by the polymer poly (vinyl pyrrolidone) (PVP) in two solvent conditions. Peptide miscibility and further self-assembling propensity in the solvents played a major role in fiber formation. When employing acetic acid as solvent, peptide fibers (0.5 µm) are formed around PVP fibers (0.75 µm), whereas in isopropanol only one type of fibers are formed, consisting of mixed peptide and PVP (1 µm). This report highlights solvent modulation as a mean to obtain different peptide sub-microfibers via a single injection nozzle in solution blow spinning. We anticipate this strategy to be applied to other small peptides with self-assembly propensity to obtain multi-functional proteinaceous fibers.

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.

Fish Gelatin-based Films for Gas Sensing.

Electronic noses (e-noses) mimic the complex biological olfactory system, usually including an array of gas sensors to act as the olfactory receptors and a trained computer with signal-processing and pattern recognition tools as the brain. In this work, a new stimuli-responsive material is shown, consisting of self-assembled droplets of liquid crystal and ionic liquid stabilised within a fish gelatin matrix. These materials change their opto/electrical properties upon contact with volatile organic compounds (VOCs). By using an in-house developed e-nose, these new gas-sensing films yield characteristic optical signals for VOCs from different chemical classes. A support vector machine classifier was implemented based on 12 features of the signals. The results show that the films are excellent identifying hydrocarbon VOCs (toluene, heptane and hexane) (95% accuracy) but lower performance was found to other VOCs, resulting in an overall 60.4% accuracy. Even though they are not reusable, these sustainable gas-sensing films are stable throughout time and reproducible, opening several opportunities for future optoelectronic devices and artificial olfaction systems.

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.

Seeing the Unseen: The Role of Liquid Crystals in Gas-Sensing Technologies.

Fast, real-time detection of gases and volatile organic compounds (VOCs) is an emerging research field relevant to most aspects of modern society, from households to health facilities, industrial units, and military environments. Sensor features such as high sensitivity, selectivity, fast response, and low energy consumption are essential. Liquid crystal (LC)-based sensors fulfill these requirements due to their chemical diversity, inherent self-assembly potential, and reversible molecular order, resulting in tunable stimuliresponsive soft materials. Sensing platforms utilizing thermotropic uniaxial systems-nematic and smectic-that exploit not only interfacial phenomena, but also changes in the LC bulk, are demonstrated. Special focus is given to the different interaction mechanisms and tuned selectivity toward gas and VOC analytes. Furthermore, the different experimental methods used to transduce the presence of chemical analytes into macroscopic signals are discussed and detailed examples are provided. Future perspectives and trends in the field, in particular the opportunities for LC-based advanced materials in artificial olfaction, are also discussed.

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.

Anything but Conventional Chromatography Approaches in Bioseparation.

While packed bed chromatography, known as conventional chromatography, has been serving the biopharmaceutical industry for decades as the bioseparation method of choice, alternative approaches are likely to take an increasing leading role in the next few years. The high number of new biological drugs under development, and the need to make biopharmaceuticals widely accessible, has been driving the academia and industry in the quest of anything but conventional chromatography approaches. In this perspective paper, these alternative approaches are discussed in view of current and future challenges in the downstream processing field.

Design and Evolution of an Opto-electronic Device for VOCs Detection.

Electronic noses (E-noses) are devices capable of detecting and identifying Volatile Organic Compounds (VOCs) in a simple and fast method. In this work, we present the development process of an opto-electronic device based on sensing films that have unique stimuli-responsive properties, altering their optical and electrical properties, when interacting with VOCs. This interaction results in optical and electrical signals that can be collected, and further processed and analysed. Two versions of the device were designed and assembled. E-nose V1 is an optical device, and E-nose V2 is a hybrid opto-electronic device. Both E-noses architectures include a delivery system, a detection chamber, and a transduction system. After the validation of the E-nose V1 prototype, the E-nose V2 was implemented, resulting in an easy-to-handle, miniaturized and stable device. Results from E-nose V2 indicated optical signals reproducibility, and the possibility of coupling the electrical signals to the optical response for VOCs sensing.

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.

A theoretical and experimental approach toward the development of affinity adsorbents for GFP and GFP-fusion proteins purification.

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.

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.