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Boronate affinity chromatography is a common purification method used for isolation and purification of cis-diol containing biomolecules. Poly (hydroxyethyl methacrylate-co-vinyl phenyl boronic acid) [P(HEMA-VPBA)] nanoparticles were prepared by miniemulsion polymerization to use in RNA purification methods. The P(HEMA-VPBA) nanoparticles were characterized by particle size distribution, surface area, Fourier transform infrared spectroscopy and atomic force microscopy. The effects of temperature, pH, RNA concentration and different salt types on RNA binding on the P(HEMA-VPBA) nanoparticles were examined. It was observed that RNA binding was increased with the increasing of pH and max RNA binding was obtained at pH 9.0. RNA binding capacity of the P(HEMA-VPBA) nanoparticles was increased from 167mg/g to 601mg/g with addition of BaCl2 to the binding medium. Maximum RNA binding capacity of the P(HEMA-VPBA) nanoparticles was 172mg/g at 1.0mg/mL initial RNA concentration. The P(HEMA-VPBA) nanoparticles were reusable for RNA binding. RNA was also extracted from Escherichia coli cells and purified successfully using the P(HEMA-VPBA) nanoparticles.

Cryogels are used effectively for many diverse applications in a variety of fields. The isolation or purification of RNA, one of the potential utilizations for cryogels, is crucial due to their vital roles such as encoding, decoding, transcription and translation, and gene expression. RNA principally exists within every living thing, but their tendency to denaturation easily is still the most challenging issue. Herein, we aimed to develop adenine incorporated polymeric cryogels as an alternative sorbent for cost-friendly and fast RNA purification with high capacity. For this goal, we synthesized the polymerizable derivative of adenine called as adenine methacrylate (AdeM) through the substitution reaction between adenine and methacryloyl chloride. Then, 2-hydroxyethyl methacrylate (HEMA)-based cryogels were prepared in a partially frozen aqueous medium by copolymerization of monomers, AdeM, and HEMA. The cryogels were characterized by using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), surface area measurements, thermogravimetric analysis (TGA), and swelling tests. RNA adsorption experiments were performed via batch system while varying different conditions including pH, initial RNA concentration, temperature, and interaction time. We achieved high RNA adsorption capacity of cryogels, with the swelling ratio around 510%, as 11.86 mg/g. The cryogels might be reused at least five times without significant decrease in adsorption capacity. © 2016 Elsevier B.V.

The purpose of this study is to synthesize megaporous cryogel loaded with hydrophobic affinity beads which can be utilized for the purification of lysozyme from chicken egg-white. N-methacryloyl-( l )-tryptophan methylester (MATrp) was used as the hydrophobic ligand. In the first step, poly(glycidyl methacrylate-N-methacryloyl-( l )-tryptophan methyl-ester) [PGMATrp] beads (2.2 μm in diameter) were produced by dispersion polymerization. In the second step, the poly(2-hydroxyethyl methacrylate) (PHEMA) cryogel loaded with PGMATrp beads [PHEMA/PGMATrp composite cryogel] was polymerized initiated by N,N,N′,N′-tetramethylene diamine (TEMED) and ammonium persulfate (APS) at −12 °C. Lysozyme adsorption capacity of the PHEMA/PGMATrp composite cryogel (332.7 mg/g polymer) was improved significantly due to the loading of PGMATrp beads into the cryogel structure. The hydrophobic MATrp comonomer played a vital role in this binding mechanism. The PHEMA/PGMATrp composite cryogel could be used many times without decreasing the lysozyme adsorption amount significantly. The main advantage of the PHEMA/PGMATrp composite cryogel is the high adsorption capacity.

WOS: 000347580500103, PubMed ID: 25454659, Molecular imprinting is a polymerization technique that provides synthetic analogs for template molecules. Molecularly imprinted polymers (MIPs) have gained much attention due to their unique properties such as selectivity and specificity for target molecules. In this study, we focused on the development of polymeric materials with molecular recognition ability, so molecular imprinting was combined with miniemulsion polymerization to synthesize self-orienting nanoparticles through the use of an epitope imprinting approach. Thus, L-lysine imprinted nanoparticles (LMIP) were synthesized via miniemulsion polymerization technique. Immunoglobulin G (IgG) was then bound to the cavities that specifically formed for L-lysine molecules that are typically found at the C-terminus of the Fc region of antibody molecules. The resulting nanoparticles makes it possible to minimize the nonspecific interaction between monomer and template molecules. In addition, the orientation of the entire IgG molecule was controlled, and random imprinting of the IgG was prevented. The optimum conditions were determined for IgG recognition using the imprinted nanoparticles. The selectivity of the nanoparticles against IgG molecules was also evaluated using albumin and hemoglobin as competitor molecules. In order to show the self-orientation capability of imprinted nanoparticles, human serum albumin (HSA) adsorption onto both the plain nanoparticles and immobilized nanoparticles by anti-human serum albumin antibody (anti-HSA antibody) was also carried out. Due to anti-HSA antibody immobilization on the imprinted nanoparticles, the adsorption capability of nanoparticles against HSA molecules vigorously enhanced. It is proved that the oriented immobilization of antibodies was appropriately succeeded

WOS: 000328593100001, PubMed ID: 23928053, In this study, we aimed to develop an alternative matrix able to deplete the albumin (Alb) and immunoglobulin G (IgG) from blood plasma simultaneously to prepare plasma samples for large-scale applications of blood-related proteomics. As a first step, nano-protein A nanoparticles (nanoProA) were prepared and characterized. Subsequently, cibacron blue F3GA (CB) was immobilized onto the nanoProA's to enhance their specific affinity for Alb molecules. Finally, both nanoparticles, specifically, nanoProA and CB-nanoProA, were separately embedded into cryogel structures to combine advantages of the nanoparticles with those of the cryogels. The protein adsorption was optimized using aqueous Alb and IgG solutions separately. Subsequently, competitive protein adsorption was performed using a protein mixture prepared with Alb and IgG adhering to their plasma protein ratios. Because of the CB-immobilization, the Alb depletion performance of the cryogels increased whereas the IgG depleting performance decreased. Using the nanoProA, embedded cryogel removed 99.3% of the IgG, while using the CB-nanoProA embedded cryogel removed 97.5% of the Alb content. The simultaneous depletion performances of the cryogels for Alb and IgG were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In this study, the monolithic cryogel-based adsorbents were classified as an alternative matrix to prepare plasma samples for proteomics applications at the preparative scale

Metal-chelating affinity beads have attracted increasing interest in recent years for protein purification. In this study, iminodiacetic acid (IDA) was covalently attached to the poly(glycidyl methacrylate) [PGMA] beads (1.6 μm in diameter). Cu(2+) ions were chelated via IDA groups on PGMA beads for affinity binding of hemoglobin (Hb) from human blood hemolysate. The PGMA beads were characterized by scanning electron microscopy (SEM). The PGMA-Cu(2+) beads (628 μmol/g) were used in the Hb binding-elution studies. The effects of Hb concentration, pH and temperature on the binding efficiency of PGMA-Cu(2+) beads were performed in a batch system. Non-specific binding of Hb to PGMA beads in the absence of Cu(2+) ions was very low (0.39 mg/g). The maximum Hb binding was 130.3 mg/g. The equilibrium Hb binding increased with increasing temperature. The negative change in Gibbs free energy (ΔG°0) indicated that the binding of Hb on the PGMA-Cu(2+) beads was a thermodynamically favorable process. The ΔS and ΔH values were 102.2 J/mol K and -2.02 kJ/mol, respectively. Significant amount of the bound Hb (up to 95.8%) was eluted in the elution medium containing 1.0 M NaCl in 1 h. The binding followed Langmuir isotherm model with monolayer binding capacity of 80.3-135.7 mg/g. Consecutive binding-elution experiments showed that the PGMA-Cu(2+) beads can be reused almost without any loss in the Hb binding capacity. To test the efficiency of Hb depletion from blood hemolysate, eluted portion was analyzed by fast protein liquid chromatography. The depletion efficiency for Hb was above 97.5%. This study determined that the PGMA-Cu(2+) beads had a superior binding capacity for Hb compared to the other carriers within this study.

In this study, phenylalanine as a hydrophobic ligand was covalently attached to the co-monomer methacrylochloride. Then, poly(2-hydroxyethylmethacrylate-co-methacrylamidophenyalanine) [poly(HEMA-MAPA)] membranes were prepared by UV-initiated photopolymerization of HEMA and methacrylamidophenyalanine. The ?-globulins adsorption onto these affinity membranes from aqueous solutions containing different amounts of ?-globulins at different pH was investigated in a batch system. The ?-globulins adsorption capacity of the membranes was increased as the ligand density on the membrane surface increase. The non-specific adsorption of the ?-globulins on the pHEMA membranes was negligible. The adsorption phenomena appeared to follow a typical Langmuir isotherm. The maximum adsorption capacity (qm) of the poly(HEMA-MAPA4) membrane for ?-globulins was 2.37 mg g-1dry membrane. The equilibrium constant (kd) value was found to be 1.61×10-1mg ml-1. More than 87% (up to 100%) of the adsorbed ?-globulins were desorbed in 120 min in the desorption medium containing 50% ethylene glycol in 1.0 M NaCl. Copyright © 2001 Elsevier Science B.V.

WOS: 000075540900001 Lysozyme adsorption onto Cibacron Blue F3GA and Cu(II) derived poly(2-hydroxyethyl methacrylate) [poly(HEMA)] membranes was investigated. Microporous poly(HEMA) membranes were prepared by photopolymerization of HEMA. The triazine dye, Cibacron Blue F3GA was attached covalently as a dye-ligand. These dye-membranes with a swelling ratio of 58% (w/w), and carrying different amounts of Cibacron Blue F3GA (between 0.35 and 1.07 mu mol cm(-2)) were used in the lysozyme adsorption studies. The effect of initial concentration and pH on the adsorption efficiency of dye-derived and metal-chelated membranes were studied in a batch reactor. The effect of Cu(II) incorporation on lysozyme adsorption was also studied. The non-specific adsorption of lysozyme on the poly(HEMA) membranes was 0.9 mu g cm(-2). Cibacron Blue F3GA attachment significantly increased the lysozyme adsorption up to 133.3 mu gcm(-2) Lysozyme adsorption capacity of the Cu(II) incorporated membranes (165.1 mu g cm(-2)) was greater than that of the Cibacron Blue F3GA-attached membranes. More than 85% of the adsorbed lysozyme was desorbed in 1 h in the desorption medium containing 0.5 M potassium thiocyanate (KSCN) at pH 8.0. (C) 1998 Elsevier Science B.V. All rights reserved.