Ionogel are versatile materials, as they present the electrical properties of ionic liquids and also dimensional stability, since they are trapped in a solid matrix, allowing application in electronic devices such as gas sensors and electronic noses. In this work, ionogels were designed to act as a sensitive layer for the detection of volatiles in a custom-made electronic nose. Ionogels composed of gelatin and a single imidazolium ionic liquid were doped with bare and functionalized iron oxide nanoparticles, producing ionogels with adjustable target selectivity. After exposing an array of four ionogels to 12 distinct volatile organic compounds, the collected signals were analyzed by principal component analysis (PCA) and by several supervised classification methods, in order to assess the ability of the electronic nose to distinguish different volatiles, which showed accuracy above 98%.

Hydrolytic zinc enzymes are common targets for protein design. The versatility of the zinc chemistry can be combined with the usage of small protein scaffolds for biocatalytic applications. Despite this, the computational design of metal-containing proteins remains challenging due to the need to properly model protein–metal interactions. We addressed these issues by developing a computational multi-state design approach of artificial zinc hydrolases based on small protein scaffolds. The zinc-finger peptide Sp1f2 was redesigned to accommodate a catalytic zinc centre and the villin headpiece C-terminal subdomain HP35 was de novo designed for metal-binding and catalytic activity. Both metallopeptides exhibited metal-induced folding (KZnP,app ≈ 2 × 105 M−1) and hydrolytic activity (k2 ≈ 0.1 M−1 s−1) towards an ester substrate. By focusing on the inherent flexibility of small proteins and their interactions with the metal ion by molecular dynamics simulations and spectroscopic studies, we identified current limitations on computational design of metalloenzymes and propose how these can be overcome by integrating information of protein–metal interactions in long time scale simulations.

Antimicrobial resistance is one of the most worrying threats to humankind with extremely high healthcare costs associated. The current technologies used in clinical microbiology to identify the bacterial agent and profile antimicrobial susceptibility are time‐consuming and frequently expensive. As a result, physicians prescribe empirical antimicrobial therapies. This scenario is often the cause of therapeutic failures, causing higher mortality rates and healthcare costs, as well as the emergence and spread of antibiotic resistant bacteria. As such, new technologies for rapid identification of the pathogen and antimicrobial susceptibility testing are needed. This review summarizes the current technologies, and the promising emerging and future alternatives for the identification and profiling of antimicrobial resistance bacterial agents, which are expected to revolutionize the field of clinical diagnostics.

Phosphoprotein-binding domains interact with cognate phosphorylated targets ruling several biological processes. The impairment of such interactions is often associated with disease development, namely cancer. The breast cancer susceptibility gene 1 (BRCA1) C-terminal (BRCT) domain is involved in the control of complex signaling networks of the DNA damage response. The capture and identification of BRCT-binding proteins and peptides may be used for the development of new diagnostic tools for diseases with abnormal phosphorylation profiles. Here we show that designed cyclic β-hairpin structures can be used as peptidomimetics of the BRCT domain, with high selectivity in binding to a target phosphorylated peptide. The amino acid residues and spatial constraints involved in the interaction between a phosphorylated peptide (GK14-P) and the BRCT domain were identified and crafted onto a 14-mer β-hairpin template in silico. Several cyclic peptides models were designed and their binding towards the target peptide and other phosphorylated peptides evaluated through virtual screening. Selected cyclic peptides were then synthesized, purified and characterized. The high affinity and selectivity of the lead cyclic peptide towards the target phosphopeptide was confirmed, and the possibility to capture it using affinity chromatography demonstrated. This work paves the way for the development of cyclic β-hairpin peptidomimetics as a novel class of affinity reagents for the highly selective identification and capture of target molecules.

As consumption of fish and fish-based foods increases, non-destructive monitoring of fish freshness also becomes more prominent. Fish products are very perishable and prone to microbiological growth, not always easily detected by organoleptic evaluation. The analysis of the headspace of fish specimens through gas sensing is an interesting approach to monitor fish freshness. Here we report a gas sensing method for monitoring Tilapia fish spoilage based on the application of a single gas sensitive gel material coupled to an optical electronic nose. The optical signals of the sensor and the extent of bacterial growth were followed over time, and results indicated good correlation between the two determinations, which suggests the potential application of this simple and low cost system for Tilapia fish freshness monitoring.

The cooperative assembly of biopolymers and small molecules can yield functional materials with precisely tunable properties. Here, the fabrication, characterization, and use of multicomponent hybrid gels as selective gas sensors are reported. The gels are composed of liquid crystal droplets self-assembled in the presence of ionic liquids, which further coassemble with biopolymers to form stable matrices. Each individual component can be varied and acts cooperatively to tune gels' structure and function. The unique molecular environment in hybrid gels is explored for supramolecular recognition of volatile compounds. Gels with distinct compositions are used as optical and electrical gas sensors, yielding a combinatorial response conceptually mimicking olfactory biological systems, and tested to distinguish volatile organic compounds and to quantify ethanol in automotive fuel. The gel response is rapid, reversible, and reproducible. These robust, versatile, modular, pliant electro-optical soft materials possess new possibilities in sensing triggered by chemical and physical stimuli.

Phosphorylation is a reversible post-translational modification of proteins that controls a plethora of cellular processes and triggers specific physiological responses, for which there is a need to develop tools to characterize phosphorylated targets efficiently. Here, a combinatorial library of triazine-based synthetic ligands comprising 64 small molecules has been rationally designed, synthesized and screened for the enrichment of phosphorylated peptides. The lead candidate (coined A8A3), composed of histidine and phenylalanine mimetic components, showed high binding capacity and selectivity for binding mono- and multi-phosphorylated peptides at pH 3. Ligand A8A3 was coupled onto both cross-linked agarose and magnetic nanoparticles, presenting higher binding capacities (100-fold higher) when immobilized on the magnetic support. The magnetic adsorbent was further screened against a tryptic digest of two phosphorylated proteins ($\alpha$- and $\beta$-caseins) and one non-phosphorylated protein (bovine serum albumin, BSA). The MALDI-TOF mass spectra of the eluted peptides allowed the identification of nine phosphopeptides, comprising both mono- and multi-phosphorylated peptides.

A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a “tag-specific” ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11 mg ml−1 and 0.48 mg ml−1 for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 106 M−1 affinity constants and Qmax values of 19.11 ± 2.60 ug g−1 and 79.39 ug g−1 for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents.

Abstract The green fluorescent protein (GFP) is a useful indicator in a broad range of applications including cell biology, gene expression and biosensing. However, its full potential is hampered by the lack of a selective, mild and low-cost purification scheme. In order to address this demand, a novel adsorbent was developed as a generic platform for the purification of \{GFP\} or \{GFP\} fusion proteins, giving \{GFP\} a dual function as reporter and purification tag. After screening a solid-phase combinatorial library of small synthetic ligands based on the Ugi-reaction, the lead ligand (A4C7) selectively recovered \{GFP\} with 94% yield and 94% purity under mild conditions and directly from Escherichia coli extracts. Adsorbents containing the ligand \{A4C7\} maintained the selectivity to recover other proteins fused to GFP. The performance of \{A4C7\} adsorbents was compared with two commercially available methods (immunoprecipitation and hydrophobic interaction chromatography), confirming the new adsorbent as a low-cost viable alternative for \{GFP\} purification.

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 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.

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.

Protein phosphorylation is a complex and highly dynamic process involved in numerous biological events. Abnormal phosphorylation is one of the underlying mechanisms for the development of cancer and metabolic disorders. The identification and absolute quantification of specific phospho-signatures can help elucidate protein functions in signaling pathways and facilitate the development of new and personalized diagnostic and therapeutic tools. This review presents a variety of strategies currently utilized for the enrichment of phosphorylated proteins and peptides before mass spectrometry analysis during proteomic studies. The investigation of specific affinity reagents, allied to the integration of different enrichment processes, is triggering the development of more selective, rapid and cost-effective high-throughput automated platforms.

This study reports the comparison of fluorimetric techniques (fluorescence microscopy and spectrofluorimetry on a 96-well format) for the on-bead screening of combinatorial libraries of affinity ligands for chromatographic separations. Two solid-phase libraries of synthetic ligands based on distinct scaffolds were synthesized by combinatorial chemistry. The libraries comprising ligands representing different hydrophobic/hydrophilic properties and sizes were tested for binding to randomly selected biomolecules (labelled with a fluorophore). Fluorescence microscopy was revealed to be a reliable and reproducible technique for the detection of lead ligands which strongly bound the target biomolecule. Results obtained by fluorescence intensity measurements in a 96-well format were less consistent, mainly due to challenges related with the accurate dispensing of the solid support.

Biomolecule separation and purification has until very recently steadfastly remained one of the more empirical aspects of modern 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.

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.