Hydroxyapatite-loaded macroporous calcium alginate hydrogels: Preparation, characterization, and in vitro evaluation.

Hydrogels from natural polysaccharides are of great interest for tissue engineering. This study aims (1) to prepare hydroxyapatite-loaded macroporous calcium alginate hydrogels by novel one-step technique using internal gelation in water-frozen solutions; (2) to evaluate their physicochemical properties; (3) to estimate their ability to support cell growth and proliferation in vitro. The structure of the hydrogel samples in a swollen state was studied by confocal laser scanning microscopy and was shown to represent a system of interconnected macropores with sizes of tens micron. The swelling behavior of the hydrogels, their mechanical properties (Young's moduli) in function of a hydroxyapatite content (5-30 mass%) were studied. All hydrogel samples loaded with hydroxyapatite were found to support growth and proliferation of mouse fibroblasts (L929) at long-term cultivation for 7 days. The obtained macroporous composite Ca-Alg-HA hydrogels could be promising for tissue engineering.

Imparting of Nearly Superparamagnetic Properties to Cryogel Scaffolds With Mesoporous MNPs for Magneto-Sensitive Tissue Engineering Strategies.

This work reports the assembly of mesoporous iron oxide nanoparticles (meso-MNPs) with cryogel scaffolds composed of chitosan and gelatin. Meso-MNPs with a particle size ranging from 2 and 50 nm, a surface area of 140.52 m2 g-1, and a pore volume of 0.27 cm3 g-1 were synthesized on a porous SiO2 template in the presence of PEG 6000 followed by leaching of SiO2. Different ratios of meso-MNPs were successfully incorporated into chitosan:gelatin cryogels up to an amount equivalent to the entire amount of polymer. The morphological structure and physicochemical properties of the cryogels were directly affected by the amount of MNPs. VSM curves showed that all composite cryogels could be magnetized by applying a magnetic field. In the context of the safety of magnetic cryogel scaffolds for use in biomedicine, it is important to note that all values are below the exposure limit for static magnetic fields, and according to cytotoxicity data, scaffolds containing meso-MNPs showed nontoxicity with cell viability ranging from 150% to 275%. In addition, microbial analysis with gram-negative and gram-positive bacteria showed that the scaffolds exhibited activity against these bacteria.

Nitric oxide-releasing photocrosslinked chitosan cryogels.

The highly porous morphology of chitosan cryogels, with submicrometric-sized pore cell walls, provides a large surface area which leads to fast water absorption and elevated swelling degrees. These characteristics are crucial for the applications of nitric oxide (NO) releasing biomaterials, in which the release of NO is triggered by the hydration of the material. In the present study, we report the development of chitosan cryogels (CS) with a porous structure of interconnected cells, with wall thicknesses in the range of 340-881 nm, capable of releasing NO triggered by the rapid hydration process. This property was obtained using an innovative strategy based on the functionalization of CS with two previously synthesized S-nitrosothiols: S-nitrosothioglycolic acid (TGA(SNO)) and S-nitrosomercaptosuccinic acid (MSA(SNO)). For this purpose, CS was previously methacrylated with glycidyl methacrylate and subsequently submitted to photocrosslinking and freeze-drying processes. The photocrosslinked hydrogels thus obtained were then functionalized with TGA(SNO) and MSA(SNO) in reactions mediated by carbodiimide. After functionalization, the hydrogels were frozen and freeze-dried to obtain porous S-nitrosated chitosan cryogels with high swelling capacities. Through chemiluminescence measurements, we demonstrated that CS-TGA(SNO) and CS-MSA(SNO) cryogels spontaneously release NO upon water absorption at rates of 3.34 × 10-2 nmol mg-1 min-1 and 1.27 × 10-1 nmol mg-1 min-1, respectively, opening new perspectives for the use of CS as a platform for localized NO delivery in biomedical applications.

Antimicrobial activity and nanoremediation of heavy metals using biosynthesized CS/GO/ZnO nanocomposite by Bacillus subtilis ATCC 6633 alone or immobilized in a macroporous cryogel.

BACKGROUND: The world society is still suffering greatly from waterborne infections, with developing countries bearing most of the morbidity and death burden, especially concerning young children. Moreover, microbial resistance is one of the most prevalent global problems that extends the need for self-medication and the healing period, or it may be linked to treatment failure that results in further hospitalization, higher healthcare expenses, and higher mortality rates. Thus, innovative synthesis of new antimicrobial materials is required to preserve the environment and enhance human health. RESULTS: The present study highlighted a simple and cost-effective approach to biosynthesize a chitosan/graphene oxide/zinc oxide nanocomposite (CS/GO/ZnO) alone and immobilized in a macroporous cryogel as a new antimicrobial agent. Bacillus subtilis ATCC 6633 was used as a safe and efficient bio-nano-factory during biosynthesis. The formation of CS/GO/ZnO was confirmed and characterized using different analyses including ultraviolet-visible spectroscopy (UV-Vis), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), selective area diffraction pattern (SADP), Zeta analyses, scanning electron microscope (SEM) and transmission electron microscopy (TEM). GO combined with ZnO NPs successfully and displayed an adsorption peak at 358 nm. The XRD results showed the crystalline composition of the loaded ZnO NPs on GO sheets. FTIR spectrum confirmed the presence of proteins during the synthesis which act as stabilizing and capping agents. The nanocomposite has a high negative surface charge (-32.8 ± 5.7 mV) which increases its stability. SEM and TEM showing the size of biosynthesized ZnO-NPs was in the range of 40-50 nm. The CS/GO/ZnO alone or immobilized in cryogel revealed good antimicrobial activities against B. cereus ATCC 14,579, Escherichia coli ATCC 25,922, and Candida albicans ATCC 10,231 in a dose-dependent manner. The CS/GO/ZnO cryogel revealed higher antimicrobial activity than GO/ZnO nanocomposite and standard antibiotics (amoxicillin and miconazole) with inhibition zones averages of 24.33 ± 0.12, 15.67 ± 0.03, and 17.5 ± 0.49 mm, respectively. The MIC values of the prepared nanocomposite against B. cereus, E. coli, and C. albicans were 80, 80, and 90 µg/ml compared to standard drugs (90, 120 and 150 µg/ml, respectively). According to the TEM ultrastructure studies of nanocomposite-treated microbes, treated cells had severe deformities and morphological alterations compared to the untreated cells including cell wall distortion, the separation between the cell wall and plasma membrane, vacuoles formation moreover complete cell lyses were also noted. In the cytotoxicity test of CS/GO/ZnO alone and its cryogel, there was a significant reduction (p˂0.05) in cell viability of WI-38 normal lung cell line after the concentration of 209 and 164 µg/ml, respectively. It showed the low toxic effect of the nanocomposite and its cryogel on the WI-38 line which implies its safety. In addition, water treatment with the CS/GO/ZnO cryogel decreased turbidity (0.58 NTU), total coliform (2 CFU/100 ml), fecal coliform (1 CFU/100 ml), fecal Streptococcus (2 CFU/100 ml), and heterotrophic plate counts (53 CFU/1 ml) not only in comparison with the chlorine-treated samples (1.69 NTU, 4 CFU/100 ml, 6 CFU/100 ml, 57 CFU/100 ml, and 140 CFU/1 ml, respectively) but also with the raw water samples (6.9 NTU, 10800 CFU/100 ml, 660 CFU/100 ml, 800 CFU/100 ml, and 4400 CFU/1 ml, respectively). Moreover, cryogel significantly decreased the concentration of different heavy metals, especially cobalt compared to chlorine (0.004 ppm, 0.002 ppm, and 0.001 ppm for raw water, chlorine-treated, and cryogel-treated groups, respectively) which helped in the reduction of their toxic effects. CONCLUSION: This study provides an effective, promising, safe, and alternative nanocomposite to treat different human and animal pathogenic microbes that might be used in different environmental, industrial, and medical applications.

Synthesis and characterization of polysaccharide-cryogel and its application to the electrochemical detection of DNA.

The main goal of our study is to demonstrate the applicability of the PPy-cryogel-modified electrodes for electrochemical detection of DNA. First, a polysaccharide-based cryogel was synthesized. This cryogel was then used as a template for chemical polypyrrole synthesis. This prepared polysaccharide-based conductive cryogel was used for electrochemical biosensing on DNA. Carrageenan (CG) and sodium alginate (SA) polysaccharides, which stand out as biocompatible materials, were used in cryogel synthesis. Electron transfer was accelerated by polypyrrole (PPy) synthesized in cryogel networks. A 2B pencil graphite electrode with a diameter of 2.00 mm was used as a working electrode. The prepared polysaccharide solution was dropped onto a working electrode as a support material to improve the immobilization capacity of biomolecules and frozen to complete the cryogelation step. PPy synthesis was performed on the electrodes whose cryogelation process was completed. In addition, the structures of cryogels synthesized on the electrode surface were characterized by thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Surface characterization of the modified electrodes was performed by energy-dispersive X-ray spectroscopy (EDX) analysis. Electrochemical determination of fish sperm DNA (fsDNA) was performed using a PPy-cryogel-modified electrode. The use of a porous 3D cryogel intermediate material enhanced the signal by providing a large surface area for the synthesis of PPy and increasing the biomolecule immobilization capacity. The detection limit was 0.98 µg mL-1 in the fsDNA concentration range 2.5-20 µg mL-1. The sensitivity of the DNA biosensor was estimated to 14.8 µA mM-1 cm-2. The stability of the biosensor under certain storage conditions was examined and observed to remain 66.95% up to 45 days.

The specific biopanning of single-domain antibody against haptens based on a functionalized cryogel.

Phage display technology is commonly applied for high-throughput screening of single-domain antibodies (sdAbs), and the problem of non-specific adsorption caused by carrier proteins seriously affects the biopanning of single-domain antibodies specific to haptens. In this paper, enrofloxacin (ENR)-functionalized cryogels were prepared by the ethylenediamine (EDA) and carbodiimide methods for application in the biopanning of ENR-specific phages. To improve the efficiency of biopanning, double blocking, a wash solution flow rate of 1 mL/min, and phage pre-incubation were applied to the biopanning process through single-factor experiments. Results of flat colony counting showed that the phage output of AG-ENR cryogels was 15 times higher than that of AG cryogels for the same input amount. And seven complete sequences of ENR-specific shark sdAbs were obtained by monoclonal phage ELISA and sequence alignment. All these results indicate that functionalized cryogels could be used as a novel and efficient method for phage biopanning for single-domain antibodies to haptens.

Synthesis and characterization of supermacroporous cryogel with immobilized p-aminobenzenesulfonamide as affinity ligand for the purification of lactoperoxidase from whey.

This study proposed the development of a monolithic supermacroporous affinity column for direct capture of lactoperoxidase, a glycoprotein present in milk, whey, and colostrum, with several applications due to its wide antimicrobial activity. A poly(acrylamide)-based cryogel was produced by radical co-polymerization of monomers in frozen aqueous solution and activated with p-aminobenzenesulfonamide as a ligand for specific interaction with the lactoperoxidase. The axial liquid dispersion coefficients at different liquid flow rates were determined by measuring residence time distributions using the tracer pulse-response method. The axial dispersion coefficient was low and the height equivalent to theoretical plate was not dependent on the flow velocity. The adsorptive capacity of affinity cryogel was studied as a function of flow velocity and the best condition was 0.9 cm/min. The response surface methodology was applied to optimize the capture of the enzyme, as a function of pH and salt concentration. Higher purification factor value was found at a salt concentration of 80 mmol/L and pH of 8.0 (p < 0.05). There was no influence of the variables under study on the yield (p > 0.05). The results indicated that affinity cryogel is a promising chromatography support for the use in high-throughput one-step purification of lactoperoxidase from whey.

Impact of double cryogelation process on a macroporous dye-affinity hydrogel.

Cryogels with interconnected channels allow high flow-through properties and mass transfer when dealing with complex mixtures such as non-clarified crude extracts. However, their mechanical strength can be challenged due to a large void volume inside the polymeric network. We have addressed this problem by forming a double-layer cryogel applied as a dye-affinity chromatography gel. In this study, poly(acrylamide-co-allyl glycidyl ether) cryogel was prepared at sub-zero temperature. The second layer was then prepared inside the primary cryogel under the same conditions to form a double-layer network. Cibacron Blue F3GA, a dye molecule, was immobilized on the surface of the cryogels. Bovine serum albumin was used as a model molecule to study the adsorption/elution procedure in batch and continuous modes. The maximum batch binding capacity and the dynamic binding capacity for the single-layer cryogel were 18 and 0.11, and for the double-layer cryogel were 7.5 and 0.9 mg/g of gel, respectively. However, the mechanical stability of the double-layer cryogel increased 7-fold (144 kPa). It was found that the kinetic and adsorption isotherms follow pseudo-second-order and Freundlich models, respectively. The regeneration of the columns after adsorption/elution cycles was evaluated, and no significant loss of capacity was observed after 10 cycles.

Cellulose-Based Cryogel Microspheres with Nanoporous and Controllable Wrinkled Morphologies for Rapid Hemostasis.

First-aid hemostatic agents for acute bleeding can save lives in emergency situations. However, rapid hemostasis remains challenging when uncontrolled hemorrhage occurs on lethal noncompressible and irregular wounds. Herein, cellulose-based cryogel microspheres with deliberately customized micromorphologies for ultrafast water transportation and diffusion, including the shark skin riblet-inspired wrinkled surface with low fluid drag and the hydrophilic nanoporous 3D networks, are developed to deal with the acute noncompressible bleeding within seconds. These cryogel microspheres can rapidly absorb a large amount of blood over 6 times their own weight in 10 s and form a robust barrier to seal a bleeding wound without applying pressure. Remarkably, massive bleeding from a cardiac penetrating hole is effectively stopped using the microspheres within 20 s and no blood leakage is observed after 30 min. Additionally, these microspheres could be readily removed without rebleeding and capillary thrombus, which is highly favorable to rapid hemostasis in emergency rescue.

Extended Rate Constants Distribution (RCD) Model for Sorption in Heterogeneous Systems: 4. Kinetics of Metal Ions Sorption in the Presence of Complexing Agents-Application to Cu(II) Sorption on Polyethyleneimine Cryogel from Acetate and Tartrate Solutions.

Here, we report a new version of the extended Rate Constants Distribution (RCD) model for metal ion sorption, which includes complex-formation equilibria. With the RCD-complex model, one can predict sorbent performance in the presence of complexing agents using data on metal ion sorption from ligand-free solutions and a set of coefficients for sorption rate constants of different ionic species. The RCD-complex model was applied to breakthrough curves of Cu(II) sorption from acetate and tartrate solutions on polyethyleneimine (PEI) monolith cryogel at different flow rates and ionic speciation. We have shown that, despite the lower stability of Cu(II)-acetate complex, at high flow rates, acetate has a more pronounced negative effect on sorption kinetics than tartrate. The RCD model was successfully used to predict the shape of the breakthrough curves at an arbitrary acetate concentration but failed to predict Cu(II) sorption from tartrate solutions in a broad range of ligand concentrations. Since a twofold increase in sorption capacity was observed at low tartrate concentrations, the latter fact was related to an alteration in the sorption mechanism of Cu(II)-ions, which depended on Cu(II) ionic speciation. The obtained results emphasize the importance of information about sorption kinetics of different ionic forms for the optimization of sorption filter performance in the presence of complexing agents.

Shape-Memory-Reduced Graphene/Chitosan Cryogels for Non-Compressible Wounds.

In this study, an antibacterial and shape-memory chitosan cryogel with high blood absorption and fast recovery from non-compressible wounds was prepared using a one-step method. Herein, we prepared a shape-memory-reduced graphene/chitosan (rGO-CTS) cryogel using a one-step method with a frozen mixing solution of chitosan, citric acid, dopamine, and graphene oxide, before treating it with alkaline solutions. The alkaline solution not only promoted the double cross-linking of chitosan but also induced dopamine to form polydopamine-reducing graphene oxide. Scanning electron microscope (SEM) images showed that the rGO-CTS cryogel possessed a uniform porous network structure, attributing excellent water-induced shape-memory properties. Moreover, the rGO-CTS cryogel exhibited good mechanical properties, antibacterial activity, and biocompatibility. In mouse liver trauma models, the rGO-CTS cryogel showed good blood clotting and hemostatic capabilities. Therefore, this composite cryogel has great potential as a new hemostatic material for application to non-compressible wounds.

Sulfonic Cryogels as Innovative Materials for Biotechnological Applications: Synthesis, Modification, and Biological Activity.

Polymeric hydrogels based on sulfo-containing comonomers are promising materials for biotechnological application, namely, for use as a system for delivering water and minerals during seed germination in conditions of an unstable moisture zone. In this work, cryogels based on 3-sulfopropyl methacrylate and 2-hydroxyethyl methacrylate copolymers were obtained by the cryotropic gelation method. The morphology, specific surface area, and swelling behaviors of cryogels are found to depend on the total concentration of monomers in the reaction system and the content of the gel fraction in cryogels. Cryogels formed in the presence of nanodiamonds are shown to exhibit high biological activity during the germination of Lepidium sativum L. variety Ajur seeds, which manifests itself by stimulating seed germination and a significant increase in the raw weight of sprouts. These results indicate that sulfonic cryogels have a high potential to improve seed germination and plant growth, proving that such cryogels can be used as environmentally friendly materials for agricultural applications.

Removal of Cd(II), Co(II), Cr(III), Ni(II), Pb(II) and Zn(II) ions from wastewater using polyethyleneimine (PEI) cryogels.

The removal of the target analytes, Cd(II), Co(II), Cr(III), Ni(II), Pb(II), and Zn(II) from contaminated waters was achieved using super porous polyethyleneimine (PEI) cryogels as adsorbent. The optimum values of the sample pH and contact time were determined as 4.0 and 90 min, respectively, for the removal of the analytes. The adsorption capacities of the sorbent were between 19.88 and 24.39 mgg-1 from 10 mL of 50 mgL-1 target metal ion solutions. The sorption kinetics of metal ions were fitted with the pseudo-second-order model. The adsorption isotherms of the target analytes into PEI cryogel were well-fitted to the Langmuir isotherm model as expected from the material homogeneity. The selectivity of the PEI cryogel in the presence of Na+, Ca2+, Mg2+, NO3-, K+ and Cl- ions even at high concentrations was tested, and the tolerance limits were satisfactory enough, e.g., the adsorption of the target analytes was even not affected in the presence of 2000 mgL-1 Ca2+, K+, Na+, Cl- and 5000 mgL-1 NO3- ions. The PEI cryogels were successfully utilized in different industrial wastewater samples that were spiked with a known amount of analytes. The removal of the analytes from wastewater samples was in the following ranges 91.94-99.86% for Cd(II), 89.59-99.89% for Co(II), 80.35-99.76% for Cr(III), 92.02-99.84% for Ni(II), 83.28-99.86% for Pb(II), and 82.94-98.24% for Zn(II), respectively. The presented novel removal strategy offers a selective, efficient, and easy application for target metal ions from industrial wastewater samples.

Adsorption and immobilization of ß-glucosidase from Thermoascus aurantiacus on macroporous cryogel by hydrophobic interaction.

Enzyme immobilization has been reported as a promising approach to improving parameters such as thermal stability, pH and reusability. In this study, a polyacrylamide cryogel functionalized with L-phenylalanine was prepared to be used in the adsorption of ß-glucosidase from Thermoascus aurantiacus, aiming at its separation and also its immobilization on the cryogel matrix. The enzyme was produced by solid state fermentation. First, the adsorption was studied as a function of the pH and the resulting yield (Y, %) and purification factor (PF, dimensionless) were determined (1.57-5.13 and 64.19-91.20, respectively). The PF and yield from eluate samples obtained at pH 3.0 were the highest (5.13 and 91.20, respectively). Then, ß-glucosidase was immobilized on the hydrophobic cryogel and the recovery activities (%) were determined as a function of temperature and in the presence of different saline solutions. The values ranged from 14.45 to 45.97. As expected, salt type and ionic strength affected the activity remained in the immobilized ß-glucosidase. The average bioreactor activity was 39.9 U/g of dry cryogel and its operational stability was measured, with no decrease in activity being observed during seven cycles. Kinetic parameters of free and immobilized enzyme were determined according to different models.

Recovering Phosphate from Complex Wastewater Using Macroporous Cryogel Composited Calcium Silicate Hydrate Nanoparticles.

Since currently used natural, nonrenewable phosphorus resources are estimated to be depleted in the next 30-200 years, phosphorus recovery from any phosphorus-rich residues has attracted great interest. In this study, phosphorus recovery from complex wastewater samples was investigated using continuous adsorption on cryogel column composited calcium silicate hydrate nanoparticles (CSH columns). The results showed that 99.99% of phosphate was recovered from a synthetic water sample (50 mg L-1) using a 5 cm CSH column with a 5 mL min-1 influent flow rate for 6 h while 82.82% and 97.58% of phosphate were recovered from household laundry wastewater (1.84 mg L-1) and reverse osmosis concentrate (26.46 mg L-1), respectively. The adsorption capacity decreased with an increasing flow rate but increased with increasing initial concentration and column height, and the obtained experimental data were better fitted to the Yoon-Nelson model (R2 = 0.7723-0.9643) than to the Adams-Bohart model (R2 = 0.6320-0.8899). The adsorption performance of phosphate was decreased 3.65 times in the presence of carbonate ions at a similar concentration, whereas no effect was obtained from nitrate and sulfate. The results demonstrate the potential of continuous-flow phosphate adsorption on the CSH column for the recovery of phosphate from complex wastewater samples.

Enhanced invertase binding from baker's yeast via cryogels included boronic acids.

Invertase, an industrially significant glycoenzyme, was purified from baker's yeast using poly (2-Hydroxyethyl methacrylate) [PHema-Pba] cryogels functionalized with boronic acid. At subzero temperatures, PHema-Pba cryogels were synthesized and characterized using swelling tests, scanning electron microscopy, and Fourier-transform infrared spectroscopy. The surface area of the PHema-Pba cryogels was 14 m2/g with a swelling ratio of 88.3% and macroporosity of 72%. The interconnected macropores of PHema-Pba cryogels were shown via scanning electron microscopy. Invertase binding capacity of PHema-Pba cryogel was evaluated by binding studies in different pH, temperature, and interaction time conditions and the maximum Invertase binding of PHema-Pba cryogel was found as 15.2 mg/g. and 23.7 fold Invertase purification was achieved from baker's yeast using PHema-Pba cryogels. The results show that PHema-Pba cryogels have high Invertase binding capacity and may be used as an alternative method for enzyme purification via boronate affinity systems.

Evaluating potential of tissue-engineered cryogels and chondrocyte derived exosomes in articular cartilage repair.

Treatment of articular cartilage injuries especially osteochondral tissue requires intervention of bioengineered scaffold. In this study, we investigated the potential of the tissue-engineered cryogel scaffold fabricated using cryogelation technology. Two types of cryogels viz. chitosan-gelatin-chondroitin sulfate (CGC) for articular cartilage and nano-hydroxyapatite-gelatin (HG) for subchondral bone were fabricated. Further, novel bilayer cryogel designed using single process fabrication of two layers (CGC as top layer and HG as the lower layer) was designed to mimic osteochondral unit. CGC cryogel was tested for their biocompatibility using the enzymatically isolated chondrcoytes from goat articular cartilage while HG cryogel was tested using pre-osteoblast cell line. Extracellular vesicles, specifically exosomes were isolated from the spent media of chondrocytes to validate their effect over cell proliferation and migration which are required for defect healing and infiltration respectively. These isolated exosomes were characterized and analyzed for confirming their size distribution profile and visualized morphologically using advanced microscopy techniques. For cartilage part, CGC cryogels were examined as delivery system for delivering exosomes at defect site, where 80% of release was observed in 72 h. Release of 18.7 µg chondroitin sulfate/mg cryogel was obtained in a period of one week from CGC cryogel (termed cryogel extract) which has chondroprotective effect. Further, effect of exosome concentration (10 and 20 µg/ml), CGC extract and combination of exosome and CGC extract (Exo-Ex) were assessed over the chondrocytes. In addition, in vitro scratch wound assay was performed to analyse the migration capacity over the micro-injury when treated with exosomes, cryogel extract and Exo-Ex. The overall results thus answer key questions of therapeutic potential of chondrocyte exosomes, cryogel extract in addition to potential of CGC and HG cryogel for osteochondral repair.

Extraction, purification by cation exchange supermacroporous cryogel and physico-chemical characterization of γ-conglutin from lupin seeds (Lupinus albus L.).

This study focused on the extraction, purification, and physicochemical characterization of γ-conglutin, a protein present in lupin seeds with properties of reducing blood glucose levels. Total protein was extracted with an alkaline-saline solvent, followed by isoelectric precipitation. Chromatographic purification of the precipitated fraction was performed using a cation exchange supermacroporous cryogel column. Electrophoresis of the eluted fraction from chromatography presented a single band of ∼48 kDa under non-reducing conditions (two bands of ∼30 and ∼17 kDa, under reducing conditions) confirming the success of the purification protocol. Liquid chromatography-tandem mass spectrometry analysis confirmed the identity of the protein as γ-conglutin. The purified γ-conglutin had an isoelectric point of 7.51, ß-sheets prevailing as a secondary structure, and denaturation temperature close to 68°C. The outcome of this work showed that γ-conglutin was obtained with a high degree of purity. The proposed purification protocol is simple and can be easily scaled up.

A hierarchical porous composite magnetic sorbent of reduced graphene oxide embedded in polyvinyl alcohol cryogel for solvent-assisted-solid phase extraction of polycyclic aromatic hydrocarbons.

A hierarchical porouscomposite magnetic sorbent was fabricated and applied to the dispersive solvent-assisted solid-phase extraction of five polycyclic aromatic hydrocarbons. A sorbent was first prepared by incorporating graphene oxide, calcium carbonate, and magnetite nanoparticles into a polyvinyl alcohol cryogel. The graphene oxide was converted to reduced graphene oxide using ascorbic acid and a hierarchical porous structure was produced by reacting hydrochloric acid with incorporated calcium carbonate to generate carbon dioxide bubbles which created a second network. Before extracting the target analytes, the extraction solvent was introduced into the hierarchical pore network of the sorbent. The extraction was based on the partition between the analytes and introduced extraction solvent and the adsorption of analytes on reduced graphene oxide.The extraction efficiency was enhanced through π-π and hydrophobic interactions between polycyclic aromatic hydrocarbons and reduced graphene oxide and extraction solvent. The extracted polycyclic aromatic hydrocarbons were determined by using high-performance liquid chromatography coupled with a fluorescence detector. The developed method was applied to extract polycyclic aromatic hydrocarbons in disposable diaper, coffee, and tea samples and recoveries from 84.5 to 99.4% were achieved with relative standard deviations below 7%. The developed sorbent exhibited good reproducibility and could be reused for 10 cycles.The developed sorbent exhibited good reproducibility and could be reused for 10 cycles.The developed sorbent exhibited good reproducibility and could be reused for 10 cycles.

In-syringe solid-phase extraction of polycyclic aromatic hydrocarbons using an iron-carboxylate metal-organic framework and hypercrosslinked polymer composite gelatin cryogel-modified cellulose acetate adsorbent.

Cellulose acetate fibers were modified with a gelatin cryogel adsorbent incorporating an iron-carboxylate metal-organic framework and hypercrosslinked polymer composite. The hybrid adsorption materials facilitated the adsorption ability toward polycyclic aromatic hydrocarbons and were entrapped into gelatin cryogel to be hierarchically coated on cellulose acetate fibers which helped to reduce the clogging problem of packed adsorbent. The composite adsorbent was employed as the solid phase of an in-syringe miniaturized solid-phase extraction system. The adsorbent was packed into the needle hub of a disposable syringe and used to extract and preconcentrate polycyclic aromatic hydrocarbons in water sample. The fabricated porous composite adsorbent was characterized and extraction conditions were optimized to achieve the best extraction performance. High-performance liquid chromatography was employed to separate and quantify extracted PAHs. The developed analysis method provided a linear range of 0.020-50 µg L-1 for phenanthrene and benzo(b)fluoranthene, 0.010-50 µg L-1 for pyrene, 0.0020-50 µg L-1 for benzo(a)anthracene, and 0.0050-50 µg L-1 for benzo(a)pyrene and dibenzo(a,h)anthracene. The limits of detection ranged from 0.5 to 5.0 ng L-1. Recoveries ranged from 89 to 98% with RSDs below 7%. The good stability of the adsorbent allowed up to 21 cycles of efficient extraction and desorption.