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Development in hemoglobin-based oxygen carriers (HBOCs) that may be used as alternatives to donated blood requires an extensive supply of highly pure hemoglobin (Hb) preparations. Therefore, it is essential to fabricate inexpensive, stable and highly selective absorbents for Hb purification. Molecular imprinting is an attractive technology for preparing such materials for targeted molecular recognition and rapid separations. In this case study, we developed human fetal hemoglobin (HbF)-imprinted polymer beads through the fusion of surface imprinting and Pickering emulsion polymerization. HbF was firstly covalently coupled to silica nanoparticles through its surface-exposed amino groups. The particle-supported HbF molecules were subsequently employed as templates for the synthesis of molecularly imprinted polymers (MIPs) with high selectivity for Hb. After removing the silica support and HbF, the resulting MIPs underwent equilibrium and kinetic binding experiments with both adult Hb (HbA) and HbF. These surface-imprinted MIPs exhibited excellent selectivity for both HbA and HbF, facilitating the one-step isolation of recombinant Hb from crude biological samples. The saturation capacities of HbA and HbF were found to be 15.4 and 17.1 mg/g polymer, respectively. The present study opens new possibilities for designed resins for tailored protein purification, separation and analysis.

Without intervention, cardiac arrhythmias pose a risk of fatality. However, timely intervention can be challenging in environments where transporting a large, heavy defibrillator is impractical, or emergency surgery to implant cardiac stimulation devices is not feasible. Here, we introduce an injectable cardiac stimulator, a syringe loaded with a nanoparticle solution comprising a conductive polymer and a monomer that, upon injection, forms a conductive structure around the heart for cardiac stimulation. Following treatment, the electrode is cleared from the body, eliminating the need for surgical extraction. The mixture adheres to the beating heart in vivo without disrupting its normal rhythm. The electrofunctionalized injectable cardiac stimulator demonstrates a tissue-compatible Young's modulus of 21 kPa and a high conductivity of 55 S/cm. The injected electrode facilitates electrocardiogram measurements, regulates heartbeat in vivo, and rectifies arrhythmia. Conductive functionality is maintained for five consecutive days, and no toxicity is observed at the organism, organ, or cellular levels., (© 2024. The Author(s).)

Proteins are adjustable units from which biomaterials with designed properties can be developed. However, non-native folded states with controlled topologies are hardly accessible in aqueous environments, limiting their prospects as building blocks. Here, we demonstrate the ability of a series of anhydrous deep eutectic solvents (DESs) to precisely control the conformational landscape of proteins. We reveal that systematic variations in the chemical composition of binary and ternary DESs dictate the stabilization of a wide range of conformations, that is, compact globular folds, intermediate folding states, or unfolded chains, as well as controlling their collective behavior. Besides, different conformational states can be visited by simply adjusting the composition of ternary DESs, allowing for the refolding of unfolded states and vice versa. Notably, we show that these intermediates can trigger the formation of supramolecular gels, also known as eutectogels, where their mechanical properties correlate to the folding state of the protein. Given the inherent vulnerability of proteins outside the native fold in aqueous environments, our findings highlight DESs as tailorable solvents capable of stabilizing various non-native conformations on demand through solvent design.

CoSAXS is a state-of-the-art SAXS/WAXS beamline exploiting the high brilliance of the MAX IV 3 GeV synchrotron. By coupling advances in sample environment control with fast X-ray detectors, millisecond time-resolved scattering methods can follow structural dynamics of proteins in solution. In the present work, four sample environments are discussed. A sample environment for combined SAXS with UV-vis and fluorescence spectroscopy (SUrF) enables a comprehensive understanding of the time evolution of conformation in a model protein upon acid-driven denaturation. The use of microfluidic chips with SAXS allows the mapping of concentration with very small sample volumes. For highly reproducible sequences of mixing of components, it is possible using stopped-flow and SAXS to access the initial effects of mixing at 2 millisecond timescales with good signal to noise to allow structural interpretation. The intermediate structures in a protein are explored under light and temperature perturbations by using lasers to "pump" the protein and SAXS as the "probe". The methods described demonstrate that features at low q, corresponding to cooperative motions of the atoms in a protein, could be extracted at millisecond timescales, which results from CoSAXS being a highly-stable, low background, dedicated SAXS beamline., (Copyright © 2024. Published by Elsevier Inc.)

The preservation of labile biomolecules presents a major challenge in chemistry, and deep eutectic solvents (DESs) have emerged as suitable environments for this purpose. However, how the hydration of DESs impacts the behavior of proteins is often neglected. Here, we demonstrate that the amino acid environment and secondary structure of two proteins (bovine serum albumin and lysozyme) and an antibody (immunoglobulin G) in 1:2 choline chloride:glycerol and 1:2 choline chloride:urea follow a re-entrant behavior with solvent hydration. A dome-shaped transition is observed with a folded or partially folded structure at very low (<10 wt % H 2 O) and high (>40 wt % H 2 O) DES hydration, while protein unfolding increases between those regimes. Hydration also affects protein conformation and stability, as demonstrated for bovine serum albumin in hydrated 1:2 choline chloride:glycerol. In the neat DES, bovine serum albumin remains partially folded and unexpectedly undergoes unfolding and oligomerization at low water content. At intermediate hydration, the protein begins to refold and gradually retrieves the native monomer-dimer equilibrium. However, ca. 36 wt % H 2 O is required to recover the native folding fully. The half-denaturation temperature of the protein increases with decreasing hydration, but even the dilute DESs significantly enhance the thermal stability of bovine serum albumin. Also, protein unfolding can be reversed by rehydrating the sample to the high hydration regime, also recovering protein function. This correlation provides a new perspective to understanding protein behavior in hydrated DESs, where quantifying the DES hydration becomes imperative to identifying the folding and stability of proteins.

Albumin-based cryogels for capturing haemin were synthesised by crosslinking different biomolecules, bovine serum albumin (BSA) and ovalbumin (OVA). The impact of the protein and coupling agent concentrations on cryogel's mechanical properties, swelling ratios and polymerisation yields, as well as autoclaving as a post-treatment on the cryogel, were studied. We found that BSA (50 mg/ml) and the crosslinker ( N -(3-dimethylaminopropyl)- N' -ethylcarbodiimide hydrochloride, 46 mg/ml) formed a cryogel with optimum physical characteristics at a comparatively low protein concentration. The cryogel's mechanical stability was increased using a double-layer cryogel approach by crosslinking the BSA proteins at subzero temperature inside an acrylamide and hydroxyethyl methacrylate premade cryogels. Batch binding and kinetic adsorption isotherms of haemin on the cryogels were assessed to evaluate their binding capacity toward the porphyrin molecule. The results showed that single-layer cryogels (BSA and OVA) had a higher capacity (∼0.68 mg/ml gel) and higher reaction rate constant towards haemin adsorption than double-layer gels. In contrast, the double-layer cryogels had higher mechanical strength than single-layer gels. The experimental results suggested that the cryogels followed the Freundlich model and the pseudo-second-order isotherm for batch adsorption and kinetics, respectively. The interaction between haemin and the gels was studied by fluorescence quenching. We found between 1.1 and 1.6 binding sites for different cryogels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hajizadeh, Dicko and Bülow.)

How many nanoparticles can we load in a fiber? How much will leak? Underlying is the relatively new question of the "space available" in fibers for nanoparticle loading. Here, using supercritical carbon dioxide (scCO 2 ) as a carrier fluid, we explored the impregnation in four Indian silks (Mulberry, Eri, Muga, and Tasar) with five standard sizes of gold nanoparticles (5, 20, 50, 100 and 150 nm in diameter). All silks could be permanently impregnated with nanoparticles up to 150 nm in size under scCO 2 impregnation. Accompanying structural changes indicated that the amorphous silk domains reorganized to accommodate the gold NPs. The mechanism was studied in detail in degummed Mulberry silk fibers (i.e., without the sericin coating) with the 5 nm nanoparticle. The combined effects of concentration, time of impregnation, scCO 2 pressure, and temperature showed that only a narrow set of conditions allowed for permanent impregnation without deterioration of the properties of the silk fibers.