Oriented immobilized anti‐hIgG via Fc fragment‐imprinted PHEMA cryogel for IgG purification

Antibodies are used in many applications, especially as diagnostic and therapeutic agents. Among the various techniques used for the purification of antibodies, immunoaffinity chromatography is by far the most common. For this purpose, oriented immobilization of antibodies is an important step for the efficiency of purification step. In this study, F_c fragment‐imprinted poly(hydroxyethyl methacrylate) cryogel (MIP) was prepared for the oriented immobilization of anti‐hIgG for IgG purification from human plasma. Non‐imprinted poly(hydroxyethyl methacrylate) cryogel (NIP) was also prepared for random immobilization of anti‐hIgG to compare the adsorption capacities of oriented (MIP/anti‐hIgG) and random (NIP/anti‐hIgG) cryogel columns. The amount of immobilized anti‐hIgG was 19.8 mg/g for the NIP column and 23.7 mg/g for the MIP column. Although the amount of immobilized anti‐hIgG was almost the same for the NIP and MIP columns, IgG adsorption capacity was found to be three times higher than the NIP/anti‐hIgG column (29.7 mg/g) for the MIP/anti‐hIgG column (86.9 mg/g). Higher IgG adsorption capacity was observed from human plasma (up to 106.4 mg/g) with the MIP/anti‐hIgG cryogel column. Adsorbed IgG was eluted using 1.0 m NaCl with a purity of 96.7%. The results obtained here are very encouraging and showed the usability of MIP/anti‐hIgG cryogel prepared via imprinting of Fc fragments as an alternative to conventional immunoaffinity techniques for IgG purification. Copyright © 2012 John Wiley & Sons, Ltd.     

Evaluation of selective composite cryogel for bromate removal from drinking water

Bromate, which is a potential carcinogen, should be removed from drinking water to levels of less than 10 μg/L. A chitosan‐based molecularly imprinted polymer (MIP) and a sol–gel ion‐exchange double hydrous oxide (Fe₂O₃·Al₂O₃·xH₂O) adsorbent (inorganic adsorbent) were prepared for this purpose. The sorption behavior of each adsorbent including sorption kinetics, isotherms, effect of pH and selective sorption were investigated in detail. Sorption experimental results showed that the MIP adsorbents had better selectivity for bromate, even in the presence of high concentrations of nitrate, as compared to the inorganic adsorbent. It was found that pH does not affect the adsorption of bromate when using the inorganic adsorbent. Additionally, both adsorbents were immobilized in a polymeric cryogel inside plastic carriers to make them more practical for using in larger scale. Regeneration of the cryogels either containing MIP or inorganic adsorbents were carried out by 0.1 M NaOH and 0.1 M NaCl, respectively. It was found that the regenerated MIP and inorganic adsorbents could be used at least three and five times, respectively, without any loss in their sorption capacity.     

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.     

Super macroporous poly(N‐isopropyl acrylamide) cryogel for separation purpose

Polymers that can respond reversibly by changing their physical or chemical properties are recognized as stimuli‐responsive polymers. The renowned temperature‐sensitive polymer is poly(N‐isopropyl acrylamide) (p(NIPAM)), and here, homopolymeric supermacroporous p(NIPAM)) cryogel was synthesized via cryopolymerization technique at cryogenic condition (below melting point of solvent, −18°C). Then, the prepared p(NIPAM) cryogel was characterized via scanning electron microscopy, Fourier transform infrared radiation spectrometer, and thermogravimetric analyzer. The lower critical solution temperature (LCST) value of the prepared p(NIPAM) cryogel was determined from % swelling equilibrium swellings at various temperatures, 20, 25, 30, 35, 40, 45, and 50°C, respectively. Furthermore, the pore volume and porosity of p(NIPAM) cryogels were compared below and above the LCST values. Finally, the separation capability of p(NIPAM) cryogels for some molecules such as tannic acid, gallic acid, nicotine (N), and caffeine (C) was investigated at the below and above the LCST values.     

Molecularly imprinted cryogel as a pH-responsive delivery system for doxorubicin

In this study, implantable and degradable molecularly imprinted cryogel was prepared for pH-responsive delivery of doxorubicin. Cryogel discs were synthesized using amino acid-based functional monomer with HEMA and gelatin. The molecularly imprinted discs were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, degradation and swelling tests. In vitro delivery experiments were carried out in order to examine the effects of medium pH and drug content. The degree of degradation of composite cryogels was found to be 83.45±1.86% after 56 days. The release profiles of DOX from molecularly imprinted cryogel discs exhibit a biphasic delivery. It was observed that an initial burst release step from 0 to 12 h was followed by a slower and sustained release. Release rate of DOX from cryogel discs increased in more acidic conditions. Kinetic studies showed that a combination of diffusion and erosion control is mainly responsible from the general release behaviors of molecularly imprinted cryogel discs.     

Preparation of poly(hydroxyethyl methacrylate) and poly(hydroxypropyl methacrylate) latices

Hydroxyethyl methacrylate and hydroxypropyl methacrylate (both having extremely high solubilities in water) were polymerized in aqueous medium to obtain the respective polymer latices with a solid content as high as 10 wt.-%. The initial state of the polymerization is in solution rather than in dispersion, and the polymer product is sparingly soluble in the aqueous phase. The polymerization was carefully controlled to avoid forming hydrogel by using an oil soluble initiator and a mixture of sodium dodecyl sulfate and poly(vinyl alcohol). Solubilities of both monomers and polymers in water were also investigated.     

Creatinine Imprinted Photonic Crystals Hydrogel Sensor

There is a demand for simple, selective, and efficient assays for the determination of clinically important metabolites such as creatinine for healthcare. Creatinine is the by-product of muscle energy metabolism and is excreted by the kidneys. To measure creatinine in the human serum, a creatinine imprinted photonic crystal hydrogel (CIPC hydrogel) for naked-eye detection is developed. CIPC hydrogel utilizes polystyrene-based two-dimensional (2D) photonic crystal colloidal arrays (PCs-array) embedded in the polyacrylamide hydrogel containing methacrylic acid which imprinted the creatinine template. The nanocavities in the hydrogel produced after the removal of the template bind to and recognize creatinine in the serum samples. The binding is selective and specific for creatinine. The binding is observed as shrinkage of the hydrogel volume and a decrease in the particle spacing which is monitored through changes in the Debye diffraction ring diameter and a visible blue-green to blue color shift. The binding event and the mechanism are investigated by molecular dockings. The CIPC sensor demonstrates a limit of detection (LOD) of 2.45 ± 1.6 µM, a linear detection range (25–500 µM), and recovery from 85.6 % to 99.9 % in the serum samples. CIPC hydrogel is available for the rapid and quantitative onsite detection of creatinine in the human serum sample.     

Isolation of immunoglobulin G from bovine milk whey by poly(hydroxyethyl methacrylate)‐based anion‐exchange cryogel

Bovine milk whey contains several bioactive proteins such as α‐lactalbumin, β‐lactoglobulin, and immunoglobulin G (IgG). Chromatographic separation of these proteins has received much attention in the past few years. In this work, we provide a chromatographic method for the efficient isolation of IgG from bovine milk whey using a poly(2‐hydroxyethyl methacrylate)‐based anion‐exchange cryogel. The monolithic cryogel was prepared by grafting 2‐(dimethylamino) ethyl methacrylate onto the poly(2‐hydroxyethyl methacrylate)‐based cryogel matrix and then employed to separate IgG under various buffer pH and salt elution conditions. The results showed that the buffer pH and the salt concentration in the step elution have remarkable influences on the purity of IgG, while the IgG recovery depended mainly on the loading volume of whey for a given cryogel bed. High purity IgG (more than 95%) was obtained using the phosphate buffer with pH of 5.8 as the running buffer and the salt solution in as the elution liquid. With suitable loading volume of whey, the maximum IgG recovery of about 94% was observed. The present separation method is thus a potential choice for the isolation of high‐purity IgG from bovine milk whey.     

Poly(hydroxyethyl methacrylate)‐based composite cryogel with embedded macroporous cellulose beads for the separation of human serum immunoglobulin and albumin

A novel super‐macroporous monolithic composite cryogel was prepared by embedding macroporous cellulose beads into poly(hydroxyethyl methacrylate) cryogel. The cellulose beads were fabricated by using a microchannel liquid‐flow focusing and cryopolymerization method, while the composite cryogel was prepared by cryogenic radical polymerization of the hydroxyethyl methacrylate monomer with poly(ethylene glycol) diacrylate as cross‐linker together with the cellulose beads. After graft polymerization with (vinylbenzyl)trimethylammonium chloride, the composite cryogel was applied to separate immunoglobulin‐G and albumin from human serum. Immunoglobulin‐G with a mean purity of 83.2% and albumin with a purity of 98% were obtained, indicating the composite cryogel as a promising chromatographic medium in bioseparation for the isolation of important bioactive proteins like immunoglobulins and albumins.     

三聚氰胺磁性分子印迹聚合物材料的制备及性能表征

利用溶剂热法通过控制反应时间和温度制得了分散性好和磁性强的Fe3O4,并利用溶胶凝胶法制备得到包覆SiO2的磁性微球(Fe3O4@SiO2)。以三聚氰胺为模板分子,甲基丙烯酸(MAA)为单体,采用本体聚合法制备了磁性分子印迹聚合物(MMIPs)。通过静态吸附实验表明,MMIPs对三聚氰胺的饱和吸附量高达10.22μg/mg,是磁性非印迹聚合物(MNIPs)的1.62倍。粒子扩散模型、Elovich模型和动态吸附实验表明所制得的MMIPs有较好的吸附性能。     

Creatinine sensor based on a molecularly imprinted polymer-modified hanging mercury drop electrode

Molecularly imprinted polymers (MIP) have been elucidated to work as artificial receptors. In our present study, a MIP was applied as a molecular recognition element to a chemical sensor. We have constructed a creatinine sensor based on a MIP layer selective for creatinine and its differential pulse, cathodic stripping voltammetric detection (DPCSV) on a hanging mercury drop electrode (HMDE). The creatinine sensor was fabricated by the drop coating of dimethylformamide (DMF) solution of a creatinine-imprinted polymer onto the surface of HMDE. The modified-HMDE, preanodised in neutral medium at +0.4 V versus Ag/AgCl for 120 s, exhibited a marked enhancement in DPCSV current in comparison to the less anodised (≤+0.3 V) HMDE. The creatinine was preconcentrated and instantaneously oxidised in MIP layer giving DPCSV response in the concentration range of 0.0025-84.0 μg mL⁻¹ [detection limit (3σ) 1.49 ng mL⁻¹]. The sensor was found to be highly selective for creatinine without any response of interferents viz., NaCl, urea, creatine, glucose, phenylalanine, tyrosine, histidine and cytosine. The non-imprinted polymer-modified electrode did not show linear response to creatinine. The imprinting factor as high as 9.4 implies that the imprinted polymer exclusively acts as a recognition element of creatinine sensor. The proposed procedure can be used to determine creatinine in human blood serum without any preliminary treatment of the sample in an accurate, rapid and simple way.     

对-特辛基酚表面印迹聚合物的制备及吸附性能评价

以对-特辛基酚为模板、甲基丙烯酸为功能单体制备了对-特辛基酚表面印迹聚合物.采用扫描电子显微镜、紫外光谱以及红外光谱对印迹聚合物进行表征,通过平衡吸附实验对聚合物的吸附性能进行评价.结果表明:该分子印迹聚合物对对-特辛基酚具有较大的吸附容量和良好的选择性,其最大吸附量Qmax约为86.12mg/g.     

Hydrogels of poly(hydroxyethyl methacrylate) and hydroxyethyl methacrylate—glycerol monomethacry-late copolymers

Hydrophilic three-dimensional polymer networks (hydrogels) were prepared from hydroxyethyl methacrylate (HEMA) and the copolymer HEMA and glycerol monomethacrylate (GMMA). The equilibrium water content of the hydrogels in water was investigated as a function of the initial dilution of the polymerization mixture, the type of solvent, and the hydrophilicity of the polymer. The initial dilution was found to have a decisive effect on the swelling or deswelling of hydrogels after the completion of the gel formation. With relatively less hydrophilic hydrogels, there is a critical initial dilution to produce the gel which does not swell or deswell in water after the gel formation. This “isovolumic” initial dilution shifts toward a higher dilution as the hydrophilicity of the hydrogels increases; however, when hydrophilicity of the polymer rises above a certain point, gels always swell in water. Permeability of oxygen through hydrogels was also studied.     

Cysteine functionalized poly(hydroxyethyl methacrylate) monolith for heavy metal removal

The aim of this study was to investigate the performance of monoliths composed of hydroxyethyl methacrylate (HEMA) to which N-methacryloyl-(L)-cysteine methyl ester (MAC) was polymerized for removal of heavy metal ions. Poly(HEMA-MAC) monolith was produced by bulk polymerization. Poly(HEMA-MAC) monolith was characterized by FTIR and scanning electron microscopy (SEM). The poly(HEMA-MAC) monolith with a swelling ratio of 89%, and containing 69.4 μmol MAC/g were used in the adsorption studies. Adsorption capacity of the monolith for the metal ions, i.e., Cu²⁺, Cd²⁺, Zn²⁺, Hg²⁺, and Pb²⁺ were investigated in aqueous media containing different amounts of the ions (10–750 mg/L) and at different pH values (3.0–7.0). The maximum adsorption capacities of the poly(HEMA-MAC) monolith were 68.2 mg/g for Zn²⁺, 129.2 mg/g for Cu²⁺, 245.8 mg/g for Pb²⁺, 270.2 mg/g for Hg²⁺, and 284.0 mg/g for Cd²⁺. pH significantly affected the adsorption capacity of MAC incorporated monolith. The competitive adsorption capacities were 587 μmol/g for Zn²⁺, 1646 μmol/g for Cu²⁺, 687 μmol/g for Pb²⁺, 929 μmol/g for Hg²⁺, and 1993 μmol/g for Cd²⁺. The chelating monolith exhibited the following metal ion affinity sequence on molar basis: Cd²⁺ > Cu²⁺ > Hg²⁺ > Pb²⁺ > Zn²⁺. The formation constants of MAC–metal ion complexes have been investigated applying the method of Ruzic. The calculated values of stability constants were 5.28 × 10⁴ L/mol for Cd²⁺, 4.16 × 10⁴ L/mol for Cu²⁺, 2.27 × 10⁴ L/mol for Hg²⁺, 1.98 × 10⁴ L/mol for Pb²⁺, and 1.25 × 10⁴ L/mol for Zn²⁺. Stability constants were increased with increasing binding affinity. The chelating monoliths can be easily regenerated by 0.1 M HNO₃ with higher effectiveness. These features make poly(HEMA-MAC) monolith a potential adsorbent for heavy metal removal.     

High capacity binding of antibodies by poly(hydroxyethyl methacrylate) nanoparticles

Poly(hydroxyethyl methacrylate) (PHEMA) nanoparticles with an average size of 150 nm in diameter and with a poly-dispersity index of 1.171 were produced by a surfactant free emulsion polymerization. Specific surface area of the PHEMA nanoparticles was found to be 1779 m(2)/g. Reactive imidazole containing 3-(2-imidazoline-1-yl)propyl(triethoxysilane) (IMEO) was used as a pseudo-specific ligand. IMEO was attached covalently onto the nanoparticles. PHEMA-IMEO nanoparticles were used for the affinity binding of immunoglobulin-G (IgG) from human plasma. To evaluate the degree of IMEO loading, the PHEMA nanoparticles were subjected to Si analysis by using flame atomizer atomic absorption spectrometer and it was estimated as 64.5 mg/g of polymer. The nanoparticles were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). IgG binding onto the PHEMA nanoparticles was found to be 5.2 mg/g. Much higher binding values (up to 843 mg/g) were obtained for the PHEMA-IMEO nanoparticles. IgG could be repeatedly bound and eluted on PHEMA-IMEO nanoparticles without noticeable loss in the IgG binding capacity.     

Characterisation of hydroxyapatite surface modified by poly(ethylene glycol) and poly(hydroxyethyl methacrylate) grafting

Hydroxyapatite (HA) has many applications in medicine as a biocompatible and bioactive biomaterial. Numerous studies have shown that modification of the HA surface can improve its biological and chemical properties. However, little is known about the surface properties of modified materials. In this paper the influence of organic polymers: polyethylene glycol (PEG) and polyhydroxyethyl methacrylate (pHEMA) on the surface properties and surface chemistry of hydroxyapatite (HA) is presented. The surface properties of modified HA were characterised by the FT-IR, XPS, BET, and zeta potential measurements. Specific surface area was determined by BET. Infrared and XPS spectra confirmed the presence of PEG and pHEMA on the surface of HA. The BET N₂ adsorption revealed slight changes in the HA surface chemistry after grafting modification. The surface chemical properties of the HA were considered to be based on the zeta potential. The decrease in zeta potential results in the increasing stability of the modified material and also in the reduction of bacterial adhesion. The reaction for surface modification of HA is proposed and described.     

Artificial enzymes based on imprinted liquid-crystalline materials

Liquid-crystal elastomers, imprinted around indole, are assessed as artificial enzymes for the isomerisation of benzisoxazole into 2-cyano phenol. Two types of material are synthesised, tested in the catalysis and compared with non-liquid-crystal imprinted polymers: an imprinted liquid-crystalline elastomer and a semi-interpenetrated imprinted liquid-crystal network. The catalytic effect of all materials is close. However, the main benefit for the liquid-crystal elastomer is shown to be the shape memory of the material at the molecular scale, because the isomerisation kinetics are found to be identical before and after deformation of the cavities either by thermal treatment up to the isotropic state or by solvent induced swelling. This fact is related to the coupling between the order and the conformation of the polymer chains, which is fixed by the crosslinking process. On the other hand, the imprinted sites of the semi-interpenetrated imprinted liquid-crystal elastomer are shown to be almost 100 times more active than the non-imprinted sites in the catalysis. This factor is only 22 for the corresponding non-liquid-crystalline network.     

Proteinases immobilized on poly(vinyl alcohol) cryogel: novel biocatalysts for peptide synthesis in organic media

Covalent immobilization of subtilisin and thermolysin on cryogel of poly(vinyl alcohol) was carried out. The biocatalysts obtained are characterized by high stability in water and in DMF—MeCN mixtures of various compositions. The synthetic efficiency of immobilized subtilisin in the multiple iterative synthesis of the peptide Z—Ala—Ala—Leu—Phe—pNA was examined in organic mixtures of different solvent compositions. Immobilized subtilisin exhibits high synthetic activity in organic media. A series of N-acylated p-nitroanilides of tetrapeptides of the general formula Z—Ala—Ala—Xaa—Yaa—pNA (Z is benzyloxycarbonyl, Xaa = Leu, Lys, or Glu; Yaa = Phe or Asp; pNA = 4-NO₂—C₆H₄NH-) were synthesized in 70—98% yields using immobilized subtilisin as a biocatalyst without activation and protection of the ionogenic groups of polyfunctional amino acids. Immobilized thermolysin in a DMF—MeCN mixture catalyzed the formation of the peptide Z—Ala—Ala—Leu—pNA, which was obtained in 90% yield (during 1 h). It was demonstrated that the biocatalyst can be used repeatedly and that it retained activity after storage in an aqueous buffer during 6 months.     

Modulated molecular recognition by a temperature‐sensitive molecularly‐imprinted polymer

Modulated molecular recognition was achieved in a temperature‐sensitive molecularly‐imprinted polymer. Using PNIPA as the temperature‐sensitive element, the adenine‐imprinted polymer (i.e., MIP‐S) was prepared and characterized. The MIP‐S exhibited a temperature‐responsive molecular recognition behavior because of the thermal phase‐transition within the MIP‐S network. Specifically, below the transition temperature (e.g., 20 °C), the MIP‐S showed a highly specific recognition for the imprint species (adenine). However, the MIP‐S did not show any significant resolution for the imprint species (adenine) and its analogue (1‐methyladenine) above the transition temperature (e.g., 40 °C). Such temperature‐regulated recognition is comparable to a switch‐on and switch‐off process, thereby making tunable molecular recognition feasible. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 2352–2360, 2009