Monosize Poly Research Articles

Characterization of a heptapeptide‐modified microsphere for oriented antibody immobilization

Oriented immobilization of antibodies is important for the effective recognition of target antigens. In this paper, a heptapeptide ligand, HWRGWVC (HC7), was modified onto non‐porous monosized poly(glyceryl methacrylate) (pGMA) microspheres (named pGMA‐HC7) to explore the antibody immobilization behaviors. Characterization of the microspheres by particle size analyzer, scanning electron microscopy, Fourier transform infrared spectroscopy, and reversed‐phase chromatography proved the success of each fabrication step. The capacity and activity of antibody immobilization through HC7 were studied using immunoglobulin G (IgG) as a model antibody and horseradish peroxidase (HRP) as a model antigen. Additionally, IgG immobilizations on pGMA microspheres by nonspecific adsorption and covalent coupling through carbodiimide chemistry were conducted for comparison. pGMA‐HC7 exhibited an IgG adsorption capacity of 3–4 mg/g in 10 min by the specific binding of HC7 without nonspecific interactions. Notably, the ligand HC7 showed a by two orders of magnitude stronger affinity for IgG than its original hexapeptide ligand HWRGWV. Moreover, the capacity and activity of the immobilized anti‐HRP antibody on pGMA‐HC7 were 1.6‐fold and 3‐fold higher than those of the covalent coupling, respectively. The results proved the superior role of HWRGWVC in the affinity binding of antibody and the potential of pGMA‐HC7‐25 in immunoassay and immunodiagnostic applications.

Concurrent determination of two opposite phase transitions in a soft polymer nanocomposite by rheology and their theoretical evaluations

The polymerization-induced biphasic nature of an emulsion 30/70wt.% methyl methacrylate (MMA)/butyl acrylate (BA) copolymer was deeply scrutinized with different techniques as the MMA-rich/BA-rich copolymers blend. Its experimentally found upper critical solution temperature (UCST) was then confirmed by the Sanchez-Lacombe lattice fluid (SLLF) and compressible regular solution (CRS) models. Characterization of soft polymer nanocomposites (SPNs) from the aforementioned system as the matrix and different fractions of mono-size poly(styrene-co-acrylonitrile) soft nanoparticles made of 70wt.% styrene (SAN70) with dynamic mechanical thermal analysis (DMTA), atomic force microscopy (AFM), and rheometry approved their multi-phase character. Homogenization temperatures of the matrix components along with the phase separations of MMA-rich/SAN70 copolymers were concurrently determined by applying Ajji and Choplin’s mean field theory on rheological measurements as a closed loop-like phase diagram. Granular state assumption regarding the soft nanoparticles by imposing geometrical constraints on their interactions based on the generalized SLLF model led to acceptable predictions about the observed lower critical solution temperature (LCST) phase behavior.

Sequential tentacle grafting and charge modification for enhancing charge density of mono-sized beads for facilitated protein refolding and purification from inclusion bodies

We have previously found that addition of like-charged media in a refolding solution can greatly enhance the refolding of pure proteins by suppressing protein aggregation. Herein, negatively charged mono-sized microspheres with sulfonic groups were fabricated to explore the facilitating effect of like-charged media on the refolding of enhanced green fluorescent protein (EGFP) expressed as inclusion bodies (IBs). A sequential polymer-tentacle grafting and sulfonate modification strategy was developed to increase the charge density of mono-sized poly(glycidyl methacrylate) (pGMA) beads (2.4μm). Namely, GMA was first grafted onto the beads by grafting polymerization to form poly(GMA) tentacles on the pGMA beads, and then the epoxy groups on the tentacles were converted into sulfonic groups by modification with sodium sulfite. By this fabrication strategy, the charge density of the beads reached 793μmol/g, about 2.8 times higher than that modified without prior grafting of the pGMA beads (285μmol/g). The negatively charged beads of different charge densities were used for facilitating the refolding of like-charged EGFP from IBs. The refolding yield as well as refolding rate increased with increasing charge density. The anti-aggregation effects of urea and like-charged microspheres were synergetic. In addition, partial purification of EGFP was achieved because the ion-exchange adsorption led to 52% removal of positively charged contaminant proteins in the refolded solution. Finally, reusability of the tentacle beads was demonstrated by repetitive EGFP refolding and recovery cycles.

Cholesterol removal from various samples by cholesterol-imprinted monosize microsphere-embedded cryogels

Cholesterol-imprinted monosize poly(glycidyl methacrylate-N-methacryloyl-(L)-tyrosine methylester) microspheres were embedded into the poly(hydroxyethyl methacrylate) (PHEMA) cryogels and the resulting composite cryogel was used for the selective removal of cholesterol. Composite cryogels were characterized by swelling tests, multipoint BET apparatus, SEM, FTIR and elemental analysis studies. Specific surface area of the PHEMA cryogel was increased from 13 to 72.7 m2/g by embedding of microspheres. Composite cryogels removed 80% of cholesterol from homogenized milk. The maximum adsorption capacity was found as 42.7 mg/g for intestinal mimicking solution. After 20 adsorption–desorption cycles, there was no remarkable decrease in the adsorption capacity.

A double-modification strategy for enhancing charge density of mono-sized beads for facilitated refolding of like-charged protein

We have previously found that addition of like-charge media in a refolding system can greatly enhance protein refolding yield, and the media of higher charge density (q) are favorable for facilitating protein refolding. Herein, a double-modification strategy was developed to increase the charge density of mono-sized poly(glycidyl methacrylate) (PGMA) microspheres (0.8μm). The PGMA beads were firstly modified with poly(ethylenimine) of molecular weight 60,000 (PEIL) or 1200 (PEIS) and then further modified with 2-diethylaminoethylchloride (DEAE) or PEIS. The charge density and bovine serum albumin (BSA) adsorption density (QBSA) of the beads were significantly increased by the double modification. The QBSA of the double-modified beads with the highest q (824μmol/g) reached 36.3mg/g, over 35% higher than that of the single-modified beads (26.7mg/g) with the highest q (300μmol/g) that a single modification could achieve. The double-modified beads with different ligand structures and charge densities were used for facilitating the refolding of like-charged lysozyme. Lysozyme refolding yield with the single-modified beads of the highest q (300μmol/g) was 60% at a critical bead concentration (cc) of 100mg/mL. By contrast, the refolding yields with the double-modified beads of q>500μmol/g were 70%, and the refolding yields with the double-modified beads of q≥650μmol/g could even reach 70% at a cc of 40mg/mL. This indicates that the double-modified beads of high q values could enhance like-charged protein refolding more significantly than the single-modified beads at a low bead utilization. The facilitating effect of the like-charged beads was independent of the ligand structure. The beads of higher q values showed lower salt sensitivity in protein refolding, and were expected beneficial for use in refolding buffers of higher ionic strengths. The sequential modification strategy for enhancing charge density would help develop more efficient media for protein adsorption and protein refolding applications.

Emulsion Copolymerization of Methyl Methacrylate/Butyl Acrylate/Iodine System to Monosize Rubbery Nanoparticles Containing Iodine and Triiodide Mixture

Monosize poly(methyl methacrylate-co-butyl acrylate) rubbery nanoparticles, approximately 95 nm in diameter, containing iodine and triiodide with a molar ratio of 2.78 of the former to the latter, were synthesized by emulsion polymerization. Iodine and the two-monomer mixture were polymerized by sodium metabisulfite and potassium persulfate as a redox initiation system. Iodine evolution to triiodide during the copolymerization was traced by ultraviolet–visible spectroscopy. Iodine addition to the monomer mixture and its copolymerization not only reduced the copolymer average molecular weight by 30%, but also led to the formation of more MMA-enriched chains. The redox initiation system also changed 26% of initial iodine molecules into triiodide, while the rest were encapsulated as molecular iodine. During thermal annealing, the lower rate of triiodide decomposition into iodine and iodide in comparison with the iodine evaporation rate broadened both thermogravimetric and heat flow curves.

Mono-sized microspheres modified with poly(ethylenimine) facilitate the refolding of like-charged lysozyme

Three mono-sized poly(glycidyl methacrylate) (PGMA) microspheres of 0.8, 1.8 and 2.5μm in particle sizes were prepared via dispersion polymerization. Poly(ethylenimine)s (PEIs) of different molecular weights (60000 and 1200) were coupled to the PGMA bead surface to prepare cationic beads of different charge densities and ligand (cationic group) structures. The cationic microspheres were used to explore the effect of solid phase properties on like-charged lysozyme refolding. The refolding yield increased with increasing bead concentration and charge density. At low bead concentration range, it increased more significantly with bead concentration with the microspheres of higher charge density. This indicates that the microspheres of high charge density are beneficial in facilitating protein refolding. The refolding yield was independent of the cationic group structure. However, PEI of higher molecular weight was favorable in the preparation of microspheres of higher charge density. Smaller-sized particles have higher specific surface area, so they facilitated lysozyme refolding more significantly. By addition of the charged beads, about 90% refolding yield of 4mg/mL lysozyme could be achieved. The studies provided more insight into the effects of like-charged solids on protein refolding, which would help design more efficient charged media for facilitated protein refolding applications.

Preparation of monodisperse PEG hydrogel microparticles using a microfluidic flow-focusing device

A microfluidic assisted preparation method of nearly monosized poly(ethylene glycol) (PEG) microparticles has been described. Three types of microfluidic flow-focusing devices with different geometries were fabricated using polydimethylsiloxane (PDMS). Microdroplets of PEG hydrogel were successfully prepared in the microfluidic flow-focusing devices by adjusting the flow rates of the continuous phase, namely, mineral oil, and the dispersed phase, viz., hydrogel solution. Then, the microdroplets of PEG hydrogel were cured by UV irradiation. Various experimental conditions pertaining to the geometry of the microfluidic flow-focusing device, flow rates of the dispersed and continuous phases, and concentration of PEG hydrogel solution were investigated and optimized to fabricate monosized PEG hydrogel microparticles. The prepared PEG microparticles were nearly monosized in the range of 40μm to 200μm in diameter according to the above experimental conditions. Then, PEG hydrogel particles laden with microbeads of 6μm diameter were fabricated using the microfluidic flow-focusing devices with the optimized conditions.

Kinetics, Isotherms and Thermodynamics of the Adsorption of Lead(II) Ions onto Porous Mono-Sized Microspheres Possessing Imidazole Functional Groups

A modified two-step seeded polymerization was used for the preparation of porous mono-sized microspheres of poly(ethylene glycol dimethacrylate-co-1-vinylimidazole) [poly(EGDMA-co-VIM)] of 7 μm in size. The specific surface area and the EGDMA/VIM molar ratio of the microspheres were determined as 64.5 m2/g and 1.5:1.0 mol/mol, respectively. The adsorption of Pb(II) ions from aqueous solution onto the porous mono-sized poly(EGDMA-co-VIM) microspheres was examined. The maximum adsorption capacities of the microspheres towards Pb(II) ions were determined as 21.74, 30.52, 49.23 and 56.48 mg/g at 293 K, 303 K, 318 K and 338 K, respectively. The dynamic and equilibrium adsorption behaviours of the system were adequately described by the pseudo-second-order kinetic model and the Langmuir isotherm, respectively. The apparent activation energy was determined as 6.212 kJ/mol, which is characteristic of a chemically controlled reaction. The values of Efe obtained from the Dubinin-Radushkevich equation in the non-lin...

Monosize microbeads for pseudo-affinity adsorption of human insulin

Affinity adsorption technique is increasingly used for protein purification, separation and other biochemical applications. Therapeutic molecules such as antibodies, cytokines, therapeutic DNA and plasma proteins must be purified before characterization and utilization. The aim of this study was to prepare micronsized spherical polymeric beads and to investigate the extent of their human insulin adsorption capability. Monosize poly(ethylene glycol dimethacrylate–N-methacryloyl-(l)-histidine) [poly(EDMA–MAH)] beads were prepared by modified suspension copolymerization. Functional monomer (MAH) was synthesized using methacryloyl chloride and l-histidine. The beads were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, swelling test and elemental analysis. MAH incorporation into monosize polymeric beads, having an average size around 2–3μm, was estimated as 55.3μmolMAH/g bead. Equilibrium swelling ratios of poly(EDMA–MAH) and poly(EDMA) beads were 65% and 55%, respectively. Adsorption experiments were performed under different conditions (i.e., pH, temperature, protein concentration and ionic strength). It was found that adsorption characteristics are strongly depend on these conditions. Maximum insulin adsorption capacity was achieved as 24.7mginsulin/g poly(EDMA–MAH) beads. Results were well fitted to the Langmuir isotherm model. Compared with poly(EDMA–MAH), nonspecific insulin adsorption onto poly(EDMA) beads was very low (0.61mginsulin/g bead) and can be negligible. It was observed that insulin could be repeatedly adsorbed and desorbed (at least 10 times) without significant loss in adsorption capacity.

Immobilized Metal Affinity Adsorption for Antibody Depletion from Human Serum with Monosize Beads

Iminodiacetic acid (IDA)-functionalized adsorbents have attracted increasing interest in recent years for immobilized metal-affinity chromatography (IMAC). In this study, IDA was covalently attached to nonporous monosize poly(glycidyl methacrylate) [poly(GMA)] beads (1.6 μm in diameter). Cu2+ ions were chelated via IDA groups for affinity depletion of immunoglobulin G (IgG) from human serum. The monosize poly(GMA) beads were characterized by scanning electron microscopy. The Cu2+-chelated beads (628 μmol/g) were used in the IgG adsorption−elution studies. Studies to determine the effects of IgG concentration, pH, and temperature on the adsorption efficiency of Cu2+-chelated beads were performed in a batch system. Nonspecific binding of IgG to monosize beads in the absence of Cu2+ ions was very low (0.45 mg/g). The IgG adsorption to chelated Cu2+ ions was 171.2 mg/g. The equilibrium IgG adsorption increased with increasing temperature. The negative change in Gibbs free energy (ΔGo < 0) indicated that the a...

Affinity adsorption of recombinant human interferon‐α on monosize dye‐affinity beads

AbstractMonosize, nonporous poly(glycidyl methacrylate) [poly(GMA)] beads were prepared by dispersion polymerization. Cibacron Blue F3GA was covalently attached onto the poly(GMA) beads for adsorption of recombinant interferon‐α (rHuIFN‐α). Monosize poly(GMA) beads were characterized by scanning electron microscopy. Dye‐carrying beads (1.73 mmol/g) were used in the adsorption–elution studies. The effect of initial concentration of rHuIFN‐α, pH, ionic strength, and temperature on the adsorption efficiency was studied in a batch system. Nonspecific adsorption of rHuIFN‐α on the beads was 0.78 mg/g. Dye attachment significantly increased the rHuIFN‐α adsorption up to 181.7 mg/g. Equilibrium adsorption of rHuIFN‐α onto the dye‐carrying beads increased with increasing temperature. Negative change in free energy (ΔG0 &lt; 0) indicated that the adsorption was a thermodynamically favorable process. ΔS and ΔH values were 146.1 J/mol K and −37.39 kJ/mol, respectively. Significant amount of the adsorbed rHuIFN‐α (up to 97.2%) was eluted in the elution medium containing 1.0M NaCl in 1 h. To determine the effects of adsorption conditions on possible conformational changes of rHuIFN‐α structure, fluorescence spectrophotometry was employed. We concluded that dye‐affinity beads can be applied for rHuIFN‐α adsorption without causing any significant conformational changes. Repeated adsorption–elution processes showed that these beads are suitable for rHuIFN‐α adsorption. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 975–981, 2007

Monosize poly(glycidyl methacrylate) beads for dye-affinity purification of lysozyme

Cibacron Blue F3GA was covalently attached onto monosize poly(glycidyl methacrylate) [poly(GMA)] beads for purification of lysozyme from chicken egg white. Monosize poly(GMA) beads, 1.6 μm in diameter, were produced by a dispersion polymerization technique. The content of epoxy groups on the surface of the poly(GMA) sample determined by the HCl–pyridine method (3.8 mmol/g). Cibacron Blue F3GA loading was 1.73 mmol/g. The monosize beads were characterized by elemental analysis, FTIR and SEM. Adsorption studies were performed under different conditions in a batch system (i.e., medium pH, protein concentration, temperature and ionic strength). Maximum lysozyme adsorption amount of poly(GMA) and poly(GMA)-Cibacron Blue F3GA beads were 1.6 and 591.7 mg/g, respectively. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium adsorption capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. It was observed that after 10 adsorption–elution cycle, poly(GMA)-Cibacron Blue F3GA beads can be used without significant loss in lysozyme adsorption capacity. Purification of lysozyme from egg-white was also investigated. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the eluted lysozyme was analyzed by SDS-PAGE and found to be 88% with recovery about 79%. The specific activity of the eluted lysozyme was high as 43,600 U/mg.

Efficient removal of albumin from human serum by monosize dye-affinity beads

Cibacron Blue F3GA was covalently attached onto monosize poly(glycidyl methacrylate) [poly(GMA)] beads for removal of human serum albumin (HSA) from human serum. Monosize poly(GMA) beads, 1.6 microm in diameter, were produced by dispersion polymerization. Cibacron Blue F3GA loading was 1.73 mol/g. HSA adsorption experiments were performed by stirred-batch adsorption. The non-specific adsorption of HSA was low (0.8 mg/g polymer). Dye attachment onto the monosize beads significantly increased the HSA adsorption (189.8 mg/g). The maximum HSA adsorption was observed at pH 5.0. With an increase of the aqueous phase concentration of sodium chloride, the adsorption capacity decreased drastically. The equilibrium adsorption of HSA significantly decreased with increasing temperature. The elution studies were performed by adding 0.1 M Tris/HCl buffer containing 0.5 M NaSCN to the HSA solutions in which adsorption equilibria had been reached. The elution results demonstrated that the adsorption of HSA to the adsorbent was reversible. The depletion efficiencies for HSA were above 87% for all studied concentrations. To test the efficiency of HSA removal from human serum, proteins in the serum and eluted portion were analyzed by two-dimensional gel electrophoresis. Eluted proteins include mainly albumin, and a small number of nonalbumin proteins such as apo-lipoprotein A1, sero-transferrin, haptoglobulin and alpha1-antitrypsin were bound by the dye-affinity beads. IgA was not identified in eluted fraction.

The effect of mono-sized liquid crystal domains on electro-optical properties in a polymer dispersed liquid crystal prepared by using monodisperse poly(methylmethacrylate)/liquid crystal microcapsules

Highly mono-sized poly(methyl methacrylate) (PMMA)/liquid crystal (LC) microcapsules having a mono-sized single LC domain were prepared by the solute codiffusion method and solvent evaporation. The size of the LC domain in the microcapsules could be controlled by the amount of LC introduced during the swelling stage. The electro-optical properties of the polymer dispersed liquid crystal (PDLC) prepared by using the microcapsules was highly improved. In particular, the threshold voltage was lowered and the switching behaviour with an applied electric field was sharpened drastically compared with PDLC prepared simply by solvent evaporation-induced phase separation.

Nonporous monosize polymeric sorbents: Dye and metal chelate affinity separation of lysozyme

Lysozyme adsorption onto dye-attached nonporous monosize poly(2-hydroxyethyl-methacrylate-methylmethacrylate) [poly(HEMA-MMA)] microspheres was investigated. Poly(HEMA-MMA) microspheres were prepared by dispersion polymerization. The monochloro-triazine dye, Cibacron Blue F3GA, was immobilized covalently as dye–ligand. These dye-affinity microspheres were used in the lysozyme adsorption–desorption studies. The effect of initial concentration of lysozyme and medium pH on the adsorption efficiency of dye-attached and metal-chelated microspheres were studied in a batch reactor. Effect of Cu(II) chelation on lysozyme adsorption was also studied. The nonspecific adsorption of lysozyme on the poly(HEMA-MMA) microspheres was 3.6 mg/g. Cibacron Blue F3GA attachment significantly increased the lysozyme adsorption up to 247.8 mg/g. Lysozyme adsorption capacity of the Cu(II) incorporated microspheres (318.9 mg/g) was greater than that of the Cibacron Blue F3GA-attached microspheres. Significant amount of the adsorbed lysozyme (up to 97%) was desorbed in 1 h in the desorption medium containing 1.0M NaSCN at pH 8.0 and 25 mM EDTA at pH 4.9. In order to examine the effects of separation conditions on possible conformational changes of lysozyme structure, fluorescence spectrophotometry was employed. We conclude that dye- and metal-chelate affinity chromatography with poly(HEMA-MMA) microspheres can be applied for lysozyme separation without causing any significant changes and denaturation. Repeated adsorption/desorption processes showed that these novel dye-attached monosize microspheres are suitable for lysozyme adsorption. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 76: 115–124, 2000

Congo red- and Zn(II)-derivatized monosize poly(MMA-HEMA) microspheres as specific sorbent in metal chelate affinity of albumin

Monosize poly(methylmethacrylate-hydroxyethylmethacrylate) [poly(MMA-HEMA)] microspheres (4 μm in diameter) were produced by dispersion copolymerization of MMA and HEMA in an ethanol-water medium. Congo Red was attached to the poly(MMA-HEMA) microspheres, covalently. These Congo Red-derivatized microspheres were characterized by optical microscopy, Fourier transform infrared spectroscopy, and elemental analysis. Then, Zn(II) ions were incorporated by chelating with the immobilized Congo Red molecules. Different amounts of Zn(II) ions [1.2–17.6 mg of Zn(II)/g of polymer] were conjugated on the microspheres by changing the initial concentration of Zn(II) ions and pH. Bovine serum albumin (BSA) adsorption on these microspheres from aqueous solutions containing different amounts of BSA at different pH and ionic strengths was investigated in batch reactors. The nonspecific BSA adsorption on the plain poly(MMA-HEMA) microspheres was very low (0.7 mg of BSA/g of polymer). Congo Red derivatization significantly increased the BSA adsorption (up to 35.8 mg of BSA/g of polymer). A further increase in the adsorption capacity (up to 61.0 mg of BSA/g of polymer) was observed when Zn(II) ions were incorporated. More than 90% of the adsorbed BSA was desorbed in 1 h in the desorption medium containing 1.0M NaSCN at pH 8.0. © 1997 John Wiley & Sons, Inc.