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Dye attached poly(hydroxyethyl methacrylate) cryogel for albumin depletion from human serum.

Cibacron Blue F3GA was immobilized on poly(hydroxyethyl methacrylate) cryogel and it was used for selective and efficient depletion of albumin from human serum. The poly(hydroxyethyl methacrylate) was selected as the basic component because of its inertness, mechanical strength, chemical and biological stability, and biocompatibility. Cibacron Blue F3GA was covalently attached to the poly(hydroxyethyl methacrylate) cryogel to produce poly(hydroxyethyl methacrylate)-Cibacron Blue F3GA cryogel affinity column. The poly(hydroxyethyl methacrylate)-Cibacron Blue F3GA cryogel was characterized with respect to gelation yield, swelling degree, total volume of macropores, Fourier Transform Infrared spectroscopy, and scanning electron microscopy. It was found that the maximum amount of adsorption (343 mg/g of dry cryogel) obtained from experimental results is very close to the calculated Langmuir adsorption capacity (345 mg/g of dry cryogel). The maximum adsorption capacity for poly(hydroxyethyl methacrylate)-Cibacron Blue F3GA cryogel column was obtained as 950 mg/g of dry cryogel for nondiluted serum. The adsorption capacity decreased with increasing dilution ratios while the depletion ratio of albumin remained as 77% in serum sample. Finally, the poly(hydroxyethyl methacrylate)-Cibacron Blue F3GA cryogel was optimized for using in the fast protein liquid chromatography system for rapid removal of the high abundant proteins from the human serum.

Affinity-recognition-based polymeric cryogels for protein depletion studies

Several affinity-recognition-based polymeric materials have been developed to aid the depletion of highly abundant proteins in serum samples. Among these, cryogels with macroporous structures may be useful in the field of protein depletion. In particular, the requirement to deplete highly abundant proteins prior to proteome investigations makes cryogels attractive for researchers. In the present review, recent developments and applications of affinity-recognition-based polymeric materials and cryogels are reviewed regarding protein depletion.

Molecularly imprinted poly(hydroxyethyl methacrylate) based cryogel for albumin depletion from human serum.

Macroporous cryogels imprinted with human serum albumin (HSA) have been prepared by copolymerization of 2-hydroxyethyl methacrylate with a functional co-monomer of N-methacryloyl-L-phenylalanine. The cryogels were used for the depletion of HSA from human serum. HSA-imprinted cryogels were prepared with gel fraction yields up to 90%, and their chemical structure, morphology and porosity were characterized by FTIR-spectroscopy, scanning electron microscopy, swelling studies and flow dynamics. Selective binding experiments were performed in the presence of competitive proteins like human transferrin and myoglobin. Albumin-imprinted cryogel column was optimized for fast protein liquid chromatography. Sodium-dodecyl sulfate polyacrylamide gel electrophoresis was used to show the efficiency of albumin depletion.

Molecularly imprinted composite cryogel for albumin depletion from human serum

A new composite protein‐imprinted macroporous cryogel was prepared for depletion of albumin from human serum prior to use in proteom applications. Polyhydroxyethyl‐methacylate‐based molecularly ımprinted polymer (MIP) composite cryogel was prepared with high gel fraction yields up to 83%, and its morphology and porosity were characterized by Fourier transform ınfrared, scanning electron microscopy, swelling studies, flow dynamics, and surface area measurements. Selective binding experiments were performed in the presence of competitive proteins human transferrin (HTR) and myoglobin (MYB). MIP composite cryogel exhibited a high binding capacity and selectivity for human serum albumin (HSA) in the presence of HTR and MYB. The competitive adsorption amount for HSA in MIP composite cryogel is 722.1 mg/dL in the presence of competitive proteins (HTR and MYB). MIP composite cryogel column was successfully applied in the fast protein liquid chromatography system for selective depletion of albumin in human serum. The depletion ratio was highly increased by embedding beads into cryogel (85%). Finally, MIP composite cryogel can be reused many times with no apparent decrease in HSA adsorption capacity. Copyright

Affinity based and molecularly imprinted cryogels: Applications in biomacromolecule purification

The publications in macro-molecularly imprinted polymers have increased drastically in recent years with the development of water-based polymer systems. The macroporous structure of cryogels has allowed the use of these materials within different applications, particularly in affinity purification and molecular imprinting based methods. Due to their high selectivity, specificity, efficient mass transfer and good reproducibility, molecularly imprinted cryogels (MICs) have become attractive for researchers in the separation and purification of proteins. In this review, the recent developments in affinity based cryogels and molecularly imprinted cryogels in protein purification are reviewed comprehensively.

Performance of dye-affinity beads for aluminium removal in magnetically stabilized fluidized bed

BackgroundAluminum has recently been recognized as a causative agent in dialysis encephalopathy, osteodystrophy, and microcytic anemia occurring in patients with chronic renal failure who undergo long-term hemodialysis. Only a small amount of Al(III) in dialysis solutions may give rise to these disorders.MethodsMagnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads in the size range of 80–120 μm were produced by free radical co-polymerization of HEMA and ethylene dimethacrylate (EDMA) in the presence of magnetite particles (Fe3O4). Then, metal complexing ligand alizarin yellow was covalently attached onto mPHEMA beads. Alizarin yellow loading was 208 μmol/g. These beads were used for the removal of Al(III) ions from tap and dialysis water in a magnetically stabilized fluidized bed.ResultsAl(III) adsorption capacity of the beads decreased with an increase in the flow-rate. The maximum Al(III) adsorption was observed at pH 5.0. Comparison of batch and magnetically stabilized fluidized bed (MSFB) maximum capacities determined using Langmuir isotherms showed that dynamic capacity (17.5 mg/g) was somewhat higher than the batch capacity (11.8 mg/g). The dissociation constants for Al(III) were determined using the Langmuir isotherm equation to be 27.3 mM (MSFB) and 6.7 mM (batch system), indicating medium affinity, which was typical for pseudospecific affinity ligands. Al(III) ions could be repeatedly adsorbed and desorbed with these beads without noticeable loss in their Al(III) adsorption capacity.ConclusionsAdsorption of Al(III) demonstrate the affinity of magnetic dye-affinity beads. The MSFB experiments allowed us to conclude that this inexpensive sorbent system may be an important alternative to the existing adsorbents in the removal of aluminium.

Molecularly imprinted composite cryogels for hemoglobin depletion from human blood

A molecularly imprinted composite cryogel (MICC) was prepared for depletion of hemoglobin from human blood prior to use in proteome applications. Poly(hydroxyethyl methacrylate) based MICC was prepared with high gel fraction yields up to 90%, and characterized by Fourier transform infrared spectrophotometer, scanning electron microscopy, swelling studies, flow dynamics and surface area measurements. MICC exhibited a high binding capacity and selectivity for hemoglobin in the presence of immunoglobulin G, albumin and myoglobin. MICC column was successfully applied in fast protein liquid chromatography system for selective depletion of hemoglobin for human blood. The depletion ratio was highly increased by embedding microspheres into the cryogel (93.2%). Finally, MICC can be reused many times with no apparent decrease in hemoglobin adsorption capacity. Copyright

Molecularly imprinted cryogel for L‐glutamic acid separation

A molecular recognition based L‐glutamic acid (L‐GLU) imprinted cryogel was prepared for L‐GLU separation via chromatographic applications. The novel functional monomer N‐methacryloyl‐(L)‐glutamic acid‐Fe3+ (MAGA‐Fe3+) was synthesized to be complex with L‐GLU. The L‐GLU imprinted cryogel was prepared by free radical polymerization under semifrozen conditions in the presence of a monomer‐template complex MAGA‐Fe3+‐L‐GLU. The binding mechanism of MAGA‐Fe3+ and L‐GLU was characterized by Fourier transform infrared (FTIR) spectroscopy in detail. FTIR analyses on the synthesized MAGA‐Fe3+‐GLU complex reveals bridging bidentate and monodentate binding modes of Fe3+ in complex with the carboxylate groups of the glutamate residues. The template L‐GLU could be reversibly detached from the cryogel to form the template cavities using a 100 mM solution of HNO3. The amount of adsorbed L‐GLU was detected using the phenyl isothiocyanate method. The L‐GLU adsorption capacity of the cryogel decreased drastically from 11.3 to 6.4 μmol g−1 as the flow rate increased from 0.5 to 4.0 mL min−1. The adsorption onto the L‐GLU imprinted cryogel was highly pH dependent due to electrostatic interaction between the L‐GLU and MAGA‐Fe3+. The PHEMAGA‐Fe3+‐GLU cryogel exhibited high selectivity to the corresponding guest amino acids (i.e., D‐GLU, L‐ASN, L‐GLN, L‐, and D‐ASP). Finally, the L‐GLU imprinted cryogel was recovered and reused many times, with no significant decrease in their adsorption capacities.

Molecular Recognition-Based Detoxification of Aluminum in Human Plasma

Molecular recognition-based Al3+-imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-L-glutamic acid) (PHEMAGA–Al3+) beads were prepared to be used in selective removal of Al3+ out of human plasma overdosed with Al3+ cations. The PHEMAGA–Al3+ beads were synthesized by suspension polymerization in the presence of a template–monomer complex (MAGA–Al3+). The specific surface area of PHEMAGA–Al3+ beads was found to be 55.6 m2/g on the average. The MAGA content in the PHEMAGA–Al3+ beads were found to be 640 μmol/g polymer. The template Al3+ cations could be reversibly detached from the matrix to form PHEMAGA–Al3+ using a 50 mM solution of EDTA. The Al3+-free PHEMAGA–Al3+ beads were then exposed to a selective separation procedure of Al3+ out of human plasma, which was implemented in a continuous system by packing the beads into a separation column (10 cm long with an inner diameter of 0.9 cm) equipped with a water jacket to control the temperature. The Al3+ adsorption capacity of the PHEMAGA–Al3+ beads decreased drastically from 0.76 mg/g polymer to 0.22 mg/g polymer as the flow rate was increased from 0.3 ml/min to 1.5 ml/min. The relative selectivity coefficients of the PHEMAGA–Al3+ beads for Al3+/Fe3+, Al3+/Cu2+ and Al3+/Zn2+ were found to be 4.49, 8.95 and 32.44 times greater than those of the non-imprinted PHEMAGA beads, respectively. FT-IR analyses on the synthesized PHEMAGA–Al3+ beads reveals monodentate and bidentate binding modes of Al3+ in complex with the carboxylate groups of the glutamate residues. Density functional theory computations at the B3LYP/6-31G(d,p) basis set suggests that structured water molecules play essential role in the stability of the monodentate binding mode in 1:1 PHEMAGA–Al3+ complexes. The PHEMAGA–Al3+ beads were recovered and reused many times, with no significant decrease in their adsorption capacities.

Poly(hydroxyethylmethacrylate‐N‐methacryloyl‐(L)‐histidine‐methyl‐ester) Based Metal‐Chelate Affinity Adsorbent for Separation of Lysozyme

Abstract Comonomer and/or metal‐chelating ligand N‐methacryloyl‐(L)‐histidine‐methylester (MAH) was synthesized by using methacryloyl chloride and L‐histidine methyl ester. Spherical beads with an average diameter of 75–125 µm were produced by suspension polymerization of 2‐hydroxyethyl methacrylate (HEMA) and MAH carried out in an aqueous dispersion medium. Poly(HEMA‐MAH) beads had a specific surface area of 18.3 m2/g. Elemental analysis of MAH for nitrogen was estimated as 895 µmol/g of polymer. Then the beads were loaded with different metal ions (i.e. Zn2+, Cu2+, Ni2+) to form the metal chelate. The effect of pH, concentration of lysozyme, and metal type on the adsorption of lysozyme to the metal‐chelated beads was examined in a batch reactor. Purification of lysozyme from egg‐white was also investigated. Maximum lysozyme adsorption capacity of poly(HEMA‐MAH) beads was found to be 8.7 mg/g at pH 7.0 in phosphate buffer. Lysozyme adsorption capacity of Zn2+, Cu2+, and Ni2+‐chelated beads was higher than that of non‐chelated beads. The maximum capacities of Ni2+, Zn2+, or Cu2+‐chelated beads were 11.5, 12.6, and 37.1 mg/g, respectively. A significant amount of the adsorbed lysozyme (up to 97%) was eluted in 1 h in the elution medium containing 25 mM EDTA at pH 4.9. Repeated adsorption‐desorption process showed that this novel metal chelated beads are suitable for lysozyme adsorption. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 80% with recovery about 75%.