Sustainable plant polyesters as substrates for optical gas sensors.

The fast and non-invasive detection of odors and volatile organic compounds (VOCs) by gas sensors and electronic noses is a growing field of interest, mostly due to a large scope of potential applications. Additional drivers for the expansion of the field include the development of alternative and sustainable sensing materials. The discovery that isolated cross-linked polymeric structures of suberin spontaneously self-assemble as a film inspired us to develop new sensing composite materials consisting of suberin and a liquid crystal (LC). Due to their stimuli-responsive and optically active nature, liquid crystals are interesting probes in gas sensing. Herein, we report the isolation and the chemical characterization of two suberin types (from cork and from potato peels) resorting to analyses of gas chromatography-mass spectrometry (GC-MS), solution nuclear magnetic resonance (NMR), and X-ray photoelectron spectroscopy (XPS). The collected data highlighted their compositional and structural differences. Cork suberin showed a higher proportion of longer aliphatic constituents and is more esterified than potato suberin. Accordingly, when casted it formed films with larger surface irregularities and a higher C/O ratio. When either type of suberin was combined with the liquid crystal 5CB, the ensuing hybrid materials showed distinctive morphological and sensing properties towards a set of 12 VOCs (comprising heptane, hexane, chloroform, toluene, dichlormethane, diethylether, ethyl acetate, acetonitrile, acetone, ethanol, methanol, and acetic acid). The optical responses generated by the materials are reversible and reproducible, showing stability for 3 weeks. The individual VOC-sensing responses of the two hybrid materials are discussed taking as basis the chemistry of each suberin type. A support vector machines (SVM) algorithm based on the features of the optical responses was implemented to assess the VOC identification ability of the materials, revealing that the two distinct suberin-based sensors complement each other, since they selectively identify distinct VOCs or VOC groups. It is expected that such new environmentally-friendly gas sensing materials derived from natural diversity can be combined in arrays to enlarge selectivity and sensing capacity.

Rational design of affinity ligands for bioseparation.

Affinity adsorbents have been the cornerstone in protein purification. The selective nature of the molecular recognition interactions established between an affinity ligands and its target provide the basis for efficient capture and isolation of proteins. The plethora of affinity adsorbents available in the market reflects the importance of affinity chromatography in the bioseparation industry. Ligand discovery relies on the implementation of rational design techniques, which provides the foundation for the engineering of novel affinity ligands. The main goal for the design of affinity ligands is to discover or improve functionality, such as increased stability or selectivity. However, the methodologies must adapt to the current needs, namely to the number and diversity of biologicals being developed, and the availability of new tools for big data analysis and artificial intelligence. In this review, we offer an overview on the development of affinity ligands for bioseparation, including the evolution of rational design techniques, dating back to the years of early discovery up to the current and future trends in the field.

ß-Hairpins as peptidomimetics of human phosphoprotein-binding domains.

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 ß-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 ß-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 ß-hairpin peptidomimetics as a novel class of affinity reagents for the highly selective identification and capture of target molecules.

Magnetic separations in biotechnology.

Magnetic separations are probably one of the most versatile separation processes in biotechnology as they are able to purify cells, viruses, proteins and nucleic acids directly from crude samples. The fast and gentle process in combination with its easy scale-up and automation provide unique advantages over other separation techniques. In the midst of this process are the magnetic adsorbents tailored for the envisioned target and whose complex synthesis spans over multiple fields of science. In this context, this article reviews both the synthesis and tailoring of magnetic adsorbents for bioseparations as well as their ultimate application.

Fishing human monoclonal antibodies from a CHO cell supernatant with boronic acid magnetic particles.

In this work we have evaluated the potential of boronic acid functionalized magnetic particles for the one-step capture of a human monoclonal antibody (mAb) from a Chinese hamster ovary (CHO) cell culture supernatant. For comparison, Protein A coated magnetic particles were also used. The most important factor influencing the overall process yield and product purity in boronic acid particles was found to be the binding pH. Basic pH values promoted higher purities while resulting in decreased yields due to the competing effects of molecules such as glucose and lactate present in the cell culture supernatant. After optimization, the particles were successfully used in a multi-cycle purification process of the mAb from the CHO feedstock. Boronic acid particles were able to achieve an average overall yield of 86% with 88% removal of CHO host cell proteins (HCP) when the binding was performed at pH 7.4, while at pH 8.5 these values were 58% and 97%, respectively. In both cases, genomic DNA removal was in excess of 97%. Comparatively, Protein A particles recorded an average overall yield of 80% and an HCP removal greater than 99%. The adsorption of the mAb to the boronic acid particles was shown to be mediated by strong affinity interactions. Overall, boronic acid based purification processes can offer a cost-effective alternative to Protein A as the direct capturing step from the mammalian cell culture.

Antibody-conjugated nanoparticles for therapeutic applications.

A great challenge to clinical development is the delivery of chemotherapeutic agents, known to cause severe toxic effects, directly to diseased sites which increase the therapeutic index whilst minimizing off-target side effects. Antibody-conjugated nanoparticles offer great opportunities to overcome these limitations in therapeutics. They combine the advantages given by the nanoparticles with the ability to bind to their target with high affinity and improve cell penetration given by the antibodies. This specialized vehicle, that can encapsulate several chemotherapeutic agents, can be engineered to possess the desirable properties, allowing overcoming the successive physiological conditions and to cross biological barriers and reach a specific tissue or cell. Moreover, antibody-conjugated nanoparticles have shown the ability to be internalized through receptor-mediated endocytosis and accumulate in cells without being recognized by the P-glycoprotein, one of the main mediators of multi-drug resistance, resulting in an increase in the intracellular concentration of drugs. Also, progress in antibody engineering has allowed the manipulation of the basic antibody structure for raising and tailoring specificity and functionality. This review explores recent developments on active drug targeting by nanoparticles functionalized with monoclonal antibodies (polymeric micelles, liposomes and polymeric nanoparticles) and summarizes the opportunities of these targeting strategies in the therapy of serious diseases (cancer, inflammatory diseases, infectious diseases, and thrombosis).

Potential of boronic acid functionalized magnetic particles in the adsorption of human antibodies under mammalian cell culture conditions.

In this work, we systematically evaluated the potential of using boronic acid functionalized magnetic particles in the capturing of human immunoglobulin G under typical mammalian cell culture conditions. For comparison, Protein A coated magnetic particles were also used. The binding pH was found to significantly influence the adsorption isotherms of boronic acid particles with the higher capacities (0.216 g IgG/g support) being observed at pH 7.4. Comparatively, this value was 0.109 g IgG/g support, for Protein A particles under the same conditions. Both particles revealed very fast adsorption kinetics with more than 70% of the maximum binding capacity being achieved in a few seconds. The effect of glucose and lactate, which are known to interact with boronic acid, was evaluated. For glucose, the binding capacity was significantly influenced by the pH and decreased as pH increased. At pH 9.5, a 70% lower binding capacity was observed for glucose concentrations as low as 0.5 g/l. The effect of lactate was less pronounced and almost pH independent reaching at most 20% decrease in binding capacity. Nevertheless, the effect of both molecules was always lower at pH 7.4. The optimization of the elution conditions enabled complete recovery of bound IgG from boronic acid particles using 50mM Tris-HCl, 200 mM sorbitol, 200 mM NaCl at pH 8.5.

A biotechnological perspective on the application of iron oxide magnetic colloids modified with polysaccharides.

Iron oxide magnetic nanoparticles (MNPs) alone are suitable for a broad spectrum of applications, but the low stability and heterogeneous size distribution in aqueous medium represent major setbacks. These setbacks can however be reduced or diminished through the coating of MNPs with various polymers, especially biopolymers such as polysaccharides. Polysaccharides are biocompatible, non-toxic and renewable; in addition, they possess chemical groups that permit further functionalization of the MNPs. Multifunctional entities can be created through decoration with specific molecules e.g. proteins, peptides, drugs, antibodies, biomimetic ligands, transfection agents, cells, and other ligands. This development opens a whole range of applications for iron oxide nanoparticles. In this review the properties of magnetic structures composed of MNPs and several polysaccharides (Agarose, Alginate, Carrageenan, Chitosan, Dextran, Heparin, Gum Arabic, Pullulan and Starch) will be discussed, in view of their recent and future biomedical and biotechnological applications.

Anti-CD8 conjugated nanoparticles to target mammalian cells expressing CD8.

This work aimed at the development of targeted drug delivery systems using nanoparticles fused with antibodies. The antibody anti-human CD8 was coupled onto PLGA nanoparticles, and the ability of these particles to specifically target cells expressing CD8 was studied. The obtained particles were found to be of spherical shape exhibiting a size between 350 and 600 nm. In vitro experiments with different cellular cultures (TE671, CHO and HEK293) using unmodified nanoparticles containing rhodamine have shown that particles were present on their surface within 48 h of incubation. In vitro tests using anti-CD8 conjugated nanoparticles in CHO cell cultures indicated that all transfected cells which express CD8 show these particles on their surface within 1h of incubation. These results demonstrated that, in a shorter time, the produced particles can target cells expressing CD8 on their surface which offers the ability to reduce drug side effects. The antibody-coupled nanoparticles represent a promising approach to improve the efficacy of active targeting for lymphoblastic leukaemia therapy.

Gum Arabic coated magnetic nanoparticles with affinity ligands specific for antibodies.

A novel magnetic support based on gum Arabic (GA) coated iron oxide magnetic nanoparticles (MNP) has been endowed with affinity properties towards immunoglobulin G (IgG) molecules. The success of the in situ triazine ligand synthesis was confirmed by fluorescence assays. Two synthetic ligands previously developed for binding to IgG, named as ligand 22/8 (artificial Protein A) and ligand 8/7 (artificial Protein L) were immobilized on to MNPs coated with GA (MNP_GA). The dimension of the particles core was not affected by the surface functionalization with GA and triazine ligands. The hydrodynamic diameters of the magnetic supports indicate that the coupling of GA leads to the formation of larger agglomerates of particles with about 1 microm, but the introduction of the triazine ligands leads to a decrease on MNPs size. The non-functionalized MNP_GA bound 28 mg IgG/g, two times less than bare MNP (60 mg IgG/g). MNP_GA modified with ligand 22/8 bound 133 mg IgG/g support, twice higher than the value obtained for ligand 8/7 magnetic adsorbents (65 mg/g). Supports modified with ligand 22/8 were selected to study the adsorption and the elution of IgG. The adsorption of human IgG on this support followed a Langmuir behavior with a Q(máx) of 344 mg IgG/g support and K(a) of 1.5 x 10(5) M. The studies on different elution conditions indicated that although the 0.05 M citrate buffer (pH 3) presented good recovery yields (elution 64% of bound protein), there was occurrence of iron leaching at this acidic pH. Therefore, a potential alternative would be to elute bound protein with a 0.05 M glycine-NaOH (pH 11) buffer.

Bio-recognition and detection using liquid crystals.

Liquid crystals (LCs) are used extensively by the electronics industry as display devices. Advances in the understanding of the liquid crystalline phase and the chemistry therein lead to the development of LC exhibiting faster switching speed with greater twist angle. This in turn lead to the emergence of liquid crystal displays, rendering dial-and-needle based displays (such as those used in various meters) and cathode ray tubes obsolete. In this article, we review the history of LC and their emergence as an invaluable material for display devices and the more recent discovery of their use as sensing elements in biosensors. This new application of LC as tools in the development of fast and simple biosensors is envisaged to gain more importance in the foreseeable future.

Antibody immobilization on magnetic particles.

Magnetic particles (MNPs) offer attractive possibilities in biotechnology. MNPs can get close to a target biological entity, as their controllable sizes range from a few nanometres up to tens of nanometres, and their surface can be modified to add affinity and specificity towards desired molecules. Additionally, they can be manipulated by an external magnetic field gradient. In this work, the study of ferric oxide (Fe3O4) MNPs with different coating agents was conducted, particularly in terms of strategies for antibody attachment at the surfaces (covalent and physical adsorption) and the effects of blocking buffer composition and incubation times on the specific and non-specific interactions observed. The considered biological model system consisted of a coating antibody (goat IgG), bovine serum albumin (BSA) as blocking agent, and a complementary antibody labelled with FITC (anti-goat IgG). The detection of antibody binding was followed by fluorescence microscopy and the intensity of the signals quantified. The ratio between the mean grey values of negative and positive controls, as well as the maximum intensity attainable in positive controls, were considered in the evaluation of the assays efficiency. The covalent immobilization of the coating antibody was more successful as opposed to protein adsorption. For covalent immobilization, silica-coated MNPs, a 5% (w/v) concentration of BSA in the blocking buffer and incubation times of 1 h produced the best results in terms of assay sensitivity. However, when conducting the assay for incubation periods of 10 min, the fluorescence signal was reduced by 44% but the assay specificity was maintained.

Studies on the molecular recognition between bioactive peptides and angiotensin-converting enzyme.

High blood pressure or hypertension is a condition affecting many individuals and represents a controllable risk factor for cardiovascular diseases such as coronary heart disease and stroke. A non-pharmacological approach to manage these includes the application of food components with antihypertensive activity. Milk protein-derived peptides have been exploited as natural hypotensive agents, namely the peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), already commercialized in functional foods as a potential alternative to synthetic drugs. These bioactive peptides inhibit in vitro and in vivo the Angiotensin I-converting enzyme (ACE), a protein with an important role in blood pressure regulation. In this work, we attempted to elucidate the possible mode of interaction between the peptides and ACE, including mechanisms of binding to the cofactor Zn2+, and further contrast this with the known mode of inhibition exerted by synthetic drugs (Captopril, Enalaprilat and Lisinopril). The bioactive peptide Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR), also known to inhibit the enzyme ACE but with a lower efficiency than VPP and IPP, was utilized in the docking studies for comparison. It was observed that the best docking poses obtained for VPP and IPP were located at the ACE catalytic site with very high resemblance to the drugs mode of interaction, including the coordination with Zn2+. As for ALPMHIR, the best docking poses were located in the narrow ACE channel outside the catalytic site, representing higher affinity energies and fewer resemblances with the interaction established by drugs.

De novo design, synthesis and screening of a combinatorial library of complementary ligands directed towards the surface of cutinase from Fusarium solani pisi.

The protein surface is the interface through which a protein molecule senses the external world. The composition of this interface, in charged, polar and/or hydrophobic residues is crucial for both the activity and stability of the protein. Protein immobilization on surfaces has been extensively explored as one of the most effective approaches for stabilization. The mechanism of stabilization, however, is still poorly understood, and usually the success of any method is more a matter of trial and error rather than the result of rational concepts. The importance of local unfolding processes in a number of biologically significant processes has been recognized and attracted increasing attention. Unfolding regions have been localized in different proteins including the recombinant cutinase from Fusarium solani pisi. The study of three structural surface regions associated with early cutinase unfolding events was the basis for the approach followed in this work. A 64-member solid-phase combinatorial library of ligands was synthesized on a triazine-substituted agarose matrix using a modified 'mix and split' procedure. The combinatorial library was assessed for binding to cutinase from Fusarium solani pisi in a biologically active form. Four lead ligands (3/5, 3/7, 4/5, 4/7) have been selected in which immobilized cutinase presented a relative activity of 30-60% as compared to the free enzyme.

Wireless excitation of quartz crystals immersed in an aqueous fluid.

A novel toroidal coil geometry able to induce remote acoustic waves in quartz crystals has been evaluated for the development of (bio)sensors. Remote acoustic generation in air was obtained for two alternative toroidal coils, with corresponding electrical impedance changes of 40 Omega for a PDMS- and 140 Omega for a ferrite-supported toroid respectively. It was found that the range of remote acoustic generation relative to the spiral coil standard was much improved, increasing the axial separation of their resonant sensing element from 0.1 mm to 20 mm, thereby allowing electromagnetic wave penetration across glass walls and fluid media to be utilised. Consideration of the transduction mechanism, along with measured cyclic changes in acoustic signal as a function of rotation, indicated that the large PDMS toroidal coil produced an asymmetric electric field. It was shown for the first time that a quartz crystal blank fully immersed in an aqueous fluid could support chemically sensitive shear acoustic standing waves that were excited and detected remotely. A signal to noise ratio of 30 ratio 1 at 20.13 MHz was achieved by placing a ferrite supported toroidal coil on the lower side of a glass beaker containing a 12 x 0.25 mm AT crystal blank and 1 mL of water. This discovery allows wireless shear acoustic wave measurements to be performed with total separation between the electronic detection system and assays undertaken in fluidic systems.