Affinity Cryogels Research Articles

Preparation of supermacroporous cryogels with improved mechanical strength for efficient purification of lysozyme from chicken egg white.

A novel, facile, and robust strategy was proposed to increase the pore size and mechanical strength of cryogels. By mixing the monomers of acrylamide and 2-hydroxyethyl methacrylate as the precursor, a monolithic copolymer cryogel with large interconnected pores and thick pore walls was prepared. Hydrogen bonding between the two monomers contributed to the entanglement and aggregation of the copolymers, thickening the pore walls and resulting in larger pore sizes. Analysis via mercury porosimetry demonstrated that the interconnected pore diameter of the copolymer cryogel ranged from 10-350 µm, which was far larger than that of the cryogels from one monomer (10-50 µm). Additionally, the thicker pore walls of the copolymer cryogel improved its mechanical strength. Affinity cryogels were prepared through covalent immobilization using Tris(hydroxymethyl)aminomethane as a coupling agent, and the affinity binding of lysozymes on Tris-cryogel was evaluated by the Langmuir isothermal adsorption with the maximum adsorption capacity of 360mg/g. Compared with that of the Tris-cryogels produced from one monomer, the copolymer Tris-cryogel exhibited higher adsorption capacity and lysozyme purity, when the chicken egg white solution flowed solely driven by gravity. This work provides a new avenue for designing and developing supermacroporous cryogels for bioseparation.

Ion Imprinted Affinity Cryogels for the Selective Adsorption Uranium in Real Samples

In this research, selective adsorption of U(VI) in aqueous solutionsin the presence of various lanthanide ions by using U(VI)-imprinted cryogel polymer was conducted. For this purpose, the prepared pHEMA-(MAH)3-U(VI) cryogel polymer by free radical polymerization method. U(VI) was desorbed with 5.0 mol/L HNO3 and thus U(VI)-imprinted were created onto p-HEMA-(MAH)3 cryogel polymer. To determine the optimum conditions, in the process of selective adsorption of U(VI) ion to U(VI)-imprinted p-HEMA-(MAH)3 cryogel polymer, some parameters such as pH, flow rate, initial U(VI) concentration were investigated. Under the optimum conditions, the maximum adsorption capacity was obtained as 74.80 mg/g. Selectivity studies were also carried out in the presence of Nd(III), La(III) and Y(III) ions using U(VI)-imprinted p-HEMA-(MAH)3 cryogel polymer. The obtained adsorption order under competitive conditions was U(VI) ˃ La(III) ˃Y(III) ˃Nd(III).

Capture of lysozyme on macroporous cryogels by hydrophobic affinity chromatography

ABSTRACT Different protocols for functionalization of cryogels with L-tryptophan were tested for the lysozyme adsorption. The effect of ionic strength and pH on the adsorptive capacity () was investigated. The morphological, thermal, and mechanical properties of the cryogels were determined. A maximum adsorption for lysozyme (58.030 mg g–1) was achieved when 5% (v/v) of glutaraldehyde (at pH 6.0) and no methanol were used during the cryogel functionalization. The hydrophobic cryogel presented a porosity of 0.666. A strong single band at 14.3 kDa was visualized in SDS-PAGE, indicating that the hydrophobic affinity cryogels was efficient for the lysozyme capture.

Preparation and Properties of Cryogel Based on Poly(2-hydroxyethyl methacrylate- co-glycidyl methacrylate).

The immobilized metal affinity cryogels based on poly(2-hydroxyethyl methacrylate- co-glycidyl methacrylate) (p(HEMA-GMA)) containing hydroxy and epoxy groups were prepared by free-radical copolymerization under cryogenic condition and then functionalized with iminodiacetic acid and chelated Cu2+, Ca2+, and Fe3+ ions to the p(HEMA-GMA) cryogel. The structures of p(HEMA-GMA) and immobilized metal-affinity cryogels were analyzed by Fourier transform infrared spectroscopy and scanning electron microscopy (SEM)-energy dispersive X-ray spectroscopy. SEM results showed that the prepared cryogels had interconnected pores with the size of 30-100 μm. The performance of water swelling into the cryogels was fitted in Fickian diffusion. The adsorption property of cryogels was influenced by the immobilized ionic type, temperature, and adsorbate. The adsorption capacity of immobilized Cu2+ cryogel (p(HEMA)-Cu2+ (0.5 M) cryogel) was the highest in comparison with that of Ca2+ and Fe3+ affinity cryogels under the same condition. The maximum adsorption capacity of p(HEMA)-Cu2+ (0.5 M) cryogel for porcine pancreatic lipase was 150.14 mg/g at a higher temperature of 35 °C, whereas for bovine serum albumin, the maximum adsorption capacity was 154.11 mg/g at a lower temperature of 25 °C. The research of thermodynamics and kinetics indicated that the mechanism of the protein adsorption process corresponded to the Langmuir model and pseudo-second-order model.

Lectin attached affinity cryogels for amyloglucosidase adsorption

Preparation of poly[ethylene glycol dimethacrylate (EGDMA)-glycidyl methacrylate (GMA)] cryogel and its usability for amyloglucosidase adsorption were investigated. Cryogels were prepared by cryocopolymerization technique and then functionalized by lectin concanavalin A (Con A). The synthesized cryogel was characterized by FTIR, SEM and EDX analysis and then used for adsorption of amyloglucosidase in a continuous flow system. The maximal amyloglucosidase adsorption efficiency of Con A attached cryogel was found to be as 30.50 mg/g cryogel. Con A modified cryogels were used repeatedly for 30 times without any significant decrease at the amyloglucosidase adsorption capacity. SDS-PAGE and activity studies confirmed that the desorbed amyloglucosidase was active and retained 90% of its initial activity after the adsorption/desorption processes.

Molecularly imprinted affinity cryogels for the selective recognition of myoglobin in blood serum

The present work demonstrates the selective recognition of myoglobin in human serum in the presence of various competitive proteins such as lysozyme, cytochrome c and hemoglobin by using molecularly imprinted affinity cryogels. The imprinted affinity cryogels towards myoglobin were prepared by using cryopolymerization technique. For this purpose, N-methacryloylamido antipyrine (MAAP)-Ce(III) and myoglobin were used as the complex functional monomer and template molecule, respectively. (MAAP)-Ce(III) complex functional in the imprinted cryogel provided a luminescence feature to the cryogel and the detection of the myoglobin was successfully achieved by UV-VIS-Near Infrared (UV-VIS-NIR) Spectroscopy. The effects of some variables such as initial myoglobin concentration, temperature, pH and ionic strength on myoglobin binding to the prepared cryogels were studied. The myoglobin binding capacity of the prepared cryogels was determined to be 68 mgg−1. Myoglobin binding to the cryogels in the presence of competitive proteins lysozyme, cytochrome c and hemoglobin were also performed. The imprinted cryogels exhibited high selectivity towards myoglobin and the obtained binding order under competitive conditions was myoglobin > lysozyme > cytochrome c > hemoglobin.

Preparation of cryogel columns for depletion of hemoglobin from human blood

In this study, we aimed to prepare the metal chelate affinity cryogels for the hemoglobin (Hb) depletion. Poly(2-hydroxyethyl methacrylate) (PHEMA) cryogels were selected as base matrix because of their blood compatibility, osmotic, chemical, and mechanical stability. Cryogels are also useful when working with the viscous samples such as blood, because of their interconnected macroporous structure. Iminodiacetic acid (IDA), the chelating agent, was covalently coupled with PHEMA cryogels after activation with the epichlorohydrin and then the Ni(II) ions were chelated to the IDA-bound cryogels. The depletion of the Hb from hemolysate was shown by SDS-PAGE.

Dye affinity cryogels for plasmid DNA purification.

The aim of this study is to prepare megaporous dye-affinity cryogel discs for the purification of plasmid DNA (pDNA) from bacterial lysate. Poly(hydroxyethyl methacrylate) [PHEMA] cryogel discs were produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) redox pair in an ice bath. Cibacron Blue F3GA was used as an affinity ligand (loading amount: 68.9μmol/g polymer). The amount of pDNA adsorbed onto the PHEMA-Cibacron Blue F3GA cryogel discs first increased and then reached a plateau value (i.e., 32.5mg/g cryogel) at 3.0mg/mL pDNA concentration. Compared with the PHEMA cryogel (0.11mg/g cryogel), the pDNA adsorption capacity of the PHEMA-Cibacron Blue F3GA cryogel (32.4mg/g polymer) was improved significantly due to the Cibacron Blue 3GA immobilization onto the polymeric matrix. pDNA adsorption amount decreased from 11.7mg/g to 1.1mg/g with the increasing of NaCl concentration. The maximum pDNA adsorption was achieved at 4°C. The overall recovery of pDNA was calculated as 90%. The PHEMA-Cibacron Blue F3GA cryogel discs could be used five times without decreasing the pDNA adsorption capacity significantly. The results show that the PHEMA-Cibacron Blue F3GA cryogel discs promise high selectivity for pDNA.

Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels

In this study, poly(2-hydroxyethyl methacrylate–glycidylmethacrylate) [poly(HEMA–GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N′-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA–GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30–50μm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA–GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA–GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94mg/g cryogel at 1.0mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH5.0 with a flow rate of 0.5mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0M NaCI at pH8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS–PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase.

Synthesis and performance of megaporous immobilized metal-ion affinity cryogels for recombinant protein capture and purification

Megaporous cryogels with metal-ion affinity functionality, which possess enhanced protein-binding ability, were synthesized and their properties were investigated. These highly porous materials (pore sizes up to 100μm) allowed the direct capture of a recombinant His6-tagged protein from a partially clarified extract. The total ligand density of the material was found to be 770μmol/g. Application of a partially clarified cell extract in order to recover a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) yielded 12mg of highly purified recombinant product per gram of adsorbent. Increased dynamic binding capacities were observed upon larger degrees of grafting, although some reduction in the quality of the system hydrodynamics was also observed. Nevertheless, these immobilized metal-ion affinity cryogels show potential for a convenient single-step purification of recombinant proteins from raw cell extracts without the need for laborious pre-chromatographic sample clean-up procedures.

Immobilization of catalase via adsorption onto metal-chelated affinity cryogels

A poly (acrylamide-allylglycidyl ether) [p(AAm-AGE)] cryogel was prepared by radical polymerization of acrylamide (AAm) and allylglycidyl ether (AGE). Cibacron Blue F3GA (CB) was covalently attached to the p(AAm-AGE) cryogel via the reaction between the chloride groups of the reactive dyes and the epoxide groups of the AGE. The CB-attached p(AAm-AGE) cryogel was chelated with Fe 3+ ions. This immobilized metal ion affinity chromatography (IMAC) cryogel carrying 25.8 ± 2.0 μmol Fe 3+ ions was used in adsorption studies to interrogate the effects of pH, protein initial concentration, flow rate, temperature and ionic strength on enzyme activity. Maximum adsorption capacities were found to be 75.7 ± 1.2 mg/g for p(AAm-AGE)–CB–Fe 3+ cryogels and 60.6 ± 1.0 mg/g for p(AAm-AGE)–CB cryogels, respectively. The adsorbed amounts of catalase per unit mass of cryogel reached a plateau value at about 1.5 mg/mL at pH 6.0. The K m values were found to be 0.73 ± 0.02 g/L for the free catalase and 0.18 ± 0.02 g/L for the immobilized catalase. The V max value of free catalase (2.0 × 10 3 U/mg enzyme) was found to be lower than that of the immobilized catalase (2.5 × 10 3 U/mg enzyme). It was also observed that the enzyme could be repeatedly adsorbed and desorbed onto the p(AAm-AGE)–CB–Fe 3+ cryogel.

Selective removal of the autoantibodies from rheumatoid arthritis patient plasma using protein A carrying affinity cryogels

Rheumatoid arthritis is a chronic, progressive, deabilitating autoimmune disease that occurs in approximately 1% of adults. Rheumatoid arthritis is characterized by chronic polyarthritis and destruction of multiple joints. In this study, IgM-antibody removal from human plasma with supermacroporous poly(hydroxyethyl methacrylate) [PHEMA] cryogel carrying protein A has been evaluated. The PHEMA cryogel was prepared by bulk polymerization which proceeds in an aqueous solution of monomer frozen inside a plastic syringe (cryo-polymerization). After thawing, the PHEMA cryogel contains a continuous matrix having interconnected macropores of 10–200 μm size. Pore volume in the PHEMA cryogel was 71.6%. Protein A molecules were covalently immobilized onto the PHEMA cryogel via cyanogen bromide (CNBr) activation. The PHEMA cryogel was contacted with blood in in vitro system for the determination of blood-compatibility. The supermacroporous structure of the PHEMA cryogel makes it possible to process blood cells without blocking the cryogel column. IgM-antibody adsorption capacity decreased significantly with the increase of the plasma flow-rate. The maximum IgM-antibody adsorption amount was 42.7 mg/g. IgM-antibody molecules could be repeatedly adsorbed and eluted without noticeable loss in the IgM-antibody adsorption amount.

Supermacroporous hydrophobic affinity cryogels for protein chromatography

Abstract N -Methacryloyl- l -tryptophan (MATrp) containing poly(2-hydroxyethyl methacrylate) based supermacroporous cryogel [PHEMATrp] was prepared for lysozyme purification form chicken egg white. MATrp was synthesized by reacting methacryloyl chloride with l -tryptophan methyl ester and provided hydrophobic functionality to the cryogel. PHEMATrp cryogel with 60–100 μm pore size was obtained by free radical polymerization of HEMA and MATrp having a specific surface area of 50 m 2 /g. PHEMATrp cryogel was characterized by swelling studies, FTIR and SEM. The equilibrium swelling ratios of the cryogels were 7.18 g H 2 O/g for PHEMA and 6.99 g H 2 O/g for PHEMATrp. Lysozyme adsorption experiments were investigated under different conditions in continuous system (i.e., medium pH, flow-rate, protein concentration, temperature, salt type). Lysozyme adsorption capacity of PHEMA and PHEMATrp cryogels from aqueous solutions was estimated as 2.9 and 46.8 mg/g (0.49 and 7.85 mg/mL), respectively. Lysozyme molecules were desorbed with 0.5 M ethylene glycol solution with 91% recovery. It was observed that PHEMATrp cryogel can be used without significant decrease in lysozyme adsorption capacity after five adsorption–desorption cycles. PHEMATrp cryogel was used for the purification of lysozyme from chicken egg white. Purity of lysozyme was estimated by SDS-PAGE. Possible denaturation of purified lysozyme was checked with fluorimetric measurements. Specific activity of the purified lysozyme was found as 43,140 U/mg using Micrococcus lysodeikticus as substrate.

The potential of polymeric cryogels in bioseparation.

This is a review discussing the production and properties of cryogels (from the Greek kappa rho iota sigma (kryos) meaning frost or ice), immobilization of ligands in cryogels and the application of affinity cryogels in bioseparation. Cryotropic gel formation proceeds in a non-frozen liquid microphase existing in the macroscopically frozen sample. Due to the cryoconcentration of gel precursors in the non-frozen liquid microphase, cryogelation is characterised by a decrease in the critical concentration of gelation and an increase in gelation rates compared with traditional gelation at temperatures above freezing point. Cryogels can be obtained through the formation of both physically and covalently cross-linked heterogeneous polymer networks. Interconnected systems of macropores and sponge-like morphology are typical for cryogels, allowing unhindered diffusion of solutes of practically any size. Most of the water present in spongy cryogels is capillary bound and can be removed mechanically by squeezing. The properties of cryogels can be regulated by the temperature of cryogelation, the time the sample is kept in a frozen state and freezing/thawing rates, by the nature of the solvent and by the use of soluble and insoluble additives. The unique macroporous morphology of cryogels, in combination with osmotic, chemical and mechanical stability, makes them attractive matrices for chromatography of large entities such as protein aggregates, membrane fragments, viruses, cell organells and even whole cells. Special attention is given to immunosorption of viruses on cryogel-based sorbents. As chromatographic materials, cryogels can be used both in bead form and as spongy cylindrical blocks (monoliths) synthesized inside the chromatographic column. The macroporous nature of cryogels is also advantageous for their application as matrices in the immobilization of biocatalysts operating in both aqueous and organic solvents. New potential applications of cryogels are discussed.