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Ochratoxin A (OTA), a group of highly toxic fungal secondary metabolites, is one of the most common mycotoxins that contaminate a wide range of agricultural products. Herein, we designed a label-free and selective surface plasmon resonance (SPR) based sensor for detection of OTA contamination in dried fig. The molecularly imprinted polymer film (MIP) was fabricated onto the SPR sensor chip by using light-initiated polymerization of N-methacryloyl-(L)-phenylalanine and 2-Hydroxyethyl methacrylate in the presence of OTA as a template. SPR chips were characterized by using contact angle, atomic force microscopy, and ellipsometry. OTA imprinted sensor was displayed a broad linear range, between 0.1 ng/mL and 20 ng/mL. The limit of detection was calculated as 0.028 ng/mL. The selectivity studies of the OTA imprinted SPR sensor was performed in the presence of aflatoxin M1, aflatoxin B1, and citrinin. The imprinting factor was found to be 2.85. The adsorption behavior of the OTA imprinted sensor was found as suitable for the Langmuir model. Results displayed that the OTA imprinted SPR sensor was successfully applied to determine OTA in dried fig samples.

In this study, we introduced a new strategy to design interface imprinted polymers for a novel aspect of molecular imprinting technique, utilization of sacrificial metal oxide particles. In the first step, bovine hemoglobin (BHb) was adsorbed on zinc oxide (ZnO) particles, which were then used to synthesize polyacrylic acid-based molecular imprinting membrane by bulk polymerization in the presence of ethylene glycol dimethacrylate as a cross-linking agent. After polymerization terminated, BHb-ZnO particles were removed to leave effective imprint sites onto the bulk polymeric network which is responsible for the formation of template orientation. The characterization of membranes was investigated by using Fourier transform infrared (FTIR), Raman spectroscopy (RS), scanning electron microscopy (SEM), surface area measurements (BET analyses) and thermogravimetric analysis (TGA). The interface molecularly imprinted membranes (iMIMs) have a relatively high specific rebinding capacity of 65.98 mg/g and excellent selectivity towards BHb with a separation factor of 6.78. The equilibrium adsorption isotherms fitted well to Langmuir isotherms (R2 = 0.9944) and the value of adsorption capability (Qmax) and equilibrium constant (b) were estimated to be 73.53 mg/g and 1.36 mg/mL for the iMIM, respectively. The kinetics of adsorption fitted best to pseudo-second order (R2 = 0.9912). The ZnO particles were used not only to ensure the preservation of the imprint cavities in the polymer network but also to lead to high template removal and better rebinding kinetics. This novel design with multiple recognition sites is quite simple and suitable for the separation of biomacromolecules.

In this study, cryogel-based molecularly imprinted composite cartridges were designed for the rapid, efficient, and selective preconcentration of benzo[ a ]pyrene (BaP) from water samples. First, a BaP-imprinted poly(2-hydroxyethyl methacrylate-N-methacryloyl-(L)-phenylalanine) composite cartridge was synthesized under semi-frozen conditions and characterized by scanning electron microscopy, elemental analysis, Fourier transform infrared spectroscopy, and swelling tests. After the optimization of preconcentration parameters, i.e., pH and initial BaP concentration, the selectivity and preconcentration efficiency, and reusability of these cartridges were also evaluated. In selectivity experiments, BaP imprinted composite cartridge exhibited binding capacities 3.09, 9.52, 8.87, and 8.77-fold higher than that of the non-imprinted composite cartridge in the presence of competitors, such as benzo[ b ]fluoranthene (BbF), benzo[ k ]fluoranthene (BkF), indeno[1,2,3- cd ]pyrene (IcdP), and 1-naphthol, respectively. The method detection limit (MDL), relative standard deviation (RSD) and preconcentration efficiency (PE) of the synthesized composite cartridge were calculated as 24.86 μg/L, 1.60%, and 349.6%, respectively.

Two-dimensional HPLC (2D-HPLC) recently has received great attention due to providing high resolving power and higher peak capacities than that of 1D-HPLC, especially dealing with a wide spectrum of sample matrices containing several components. In this work, an on-line heart-cutting two-dimensional liquid chromatography (2D-LC) method was developed using monolithic columns coupled with reversed-phase liquid chromatography (RPLC). 2D-HPLC was successfully carried out using affinity-based monolithic columns at first dimension (20 cm x 4.6 mm I.D.) followed by a Pinnacle II PAH column (50 mm x 4.6 mm I.D.) at the second dimension. Furthermore, good linearity was observed for the correlation of the benzo[a]pyrene (BaP) molecule against the peak areas (R-2 = 0.994) in the concentration range of 0.01-1.0 ng/mL in 30 min. The limit of detection and the limit of quantification were found to be 4.0 pg/mL and 12.0 pg/mL, respectively. Both the intra-day and inter-day precision at 0.01 and 0.1 ng/mL spiked concentrations were below than 2.35% RSD whereas the means of the recovery data of the BaP from the water samples were found to be in the range of 93.71-98.65%. These results demonstrate that the 2D-HPLC system, developed by the combination of the P(HEMA-MAPA) column and Pinnacle II PAH column, is reliable, stable, and well qualified in the extraction of polycyclic aromatic hydrocarbons from the water samples.

The continuous bed bioreactor systems have been used for the production of protein therapeutics, such as IgG, using immobilized enzyme in biopharmaceutical applications. We developed macroporous poly(hydroxyethyl methacrylate-co-glycidyl methacrylate) cryogel-based bioreactor matrix using sodium dodecyl sulfate as surfactans in the presence of ethylene glycol dimethacrylate as cross linking agent by bulk polymerization. The developed polyGMA immobilized bioreactor with papain enzyme was used for specific fragmentation of immunoglobulin G. The catalysis efficiency for immobilized enzyme were investigated in comparison with free enzyme. The immobilized papain displayed broad catalytic activity over a variety of conditions, with maximal activity around pH 7.0 and 70 °C. The Michaelis-Menten kinetic constant (Km), the maximum reaction velocity (Vmax), and the catalytic efficiency (kcat) for free enzyme were 0.1097 mg/mL, 29.9 mg/mL/min, and 92.01 1/min, respectively, whereas for immobilized enzyme, Km, Vmax, and kcat values were 0.1078 mg/mL, 30.53 mg/mL/min, and 94.3 1/min, respectively. In a further step, after digestion, remarkable digestion products of bioreactor, Fab and Fc fragments, produced with immobilized papain bioreactors were analyzed in two ways by SDS-PAGE and reversed-phase HPLC; it was demonstrated that papain immobilized bioreactor successfully used for the digestion of human IgG with high activity. Therefore, the polyGMA cryogel immobilized with papain exhibited a very effective matrix for the bioreactor which can be considered as an alternative bioreactor matrix with great promise in biopharmaceutical applications.

WOS: 000354965300034, In this study, we synthesised specific filtration cartridges with selective recognition sites for target molecules and used them to separate interferon alpha-2b from aqueous solutions. We combined molecular imprinting technology with cryogel to achieve specific and rapid filtration of interferon alpha-2b through the macroporous structure of a cryogel network. Recombinant interferon alpha-2b-imprinted poly(2-hydroxyethyl methacrylate-N-methacryloyl-L-tryptophan) P(HEMATrp)/alpha-2bIFN cryogels were synthesised via free-radical bulk polymerisation under partially frozen conditions. The interferon alpha-2b filtration conditions were subsequently optimised with respect to factors such as pH, initial concentration, temperature, centrifugation speed, salt concentration and type and the amount of precomplex incorporated. Selectivity experiments were conducted in respect to isoelectric point as well as size of the competitor proteins under both uncompetitive and competitive conditions. The relative selectivity coefficients of the specific filtration cartridge in respect to isoelectronic points for interferon/ IgG, interferon/HSA and interferon/insulin pairs were 3.72, 7.10 and 10.68 times greater than the coefficient of a non-imprinted [P(HEMATrp)] filtration cartridge, respectively. Similarly, the relative selectivity coefficients of the specific filtration cartridge in respect to competitor size for interferon/ lysozyme, interferon/myoglobin and interferon/carbonic anhydrase pairs were calculated as 7.58, 10.40 and 11.68 under uncompetitive conditions whereas those values under competitive conditions were calculated as 1.08, 1.05 and 1.34, respectively. The results indicated that specific filtration cartridges developed could repeatedly adsorb interferon alpha-2b with a short separation time without any significant decrease in the adsorption capacity even if competitive conditions were conducted.

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

In this study cryogels based 2-hydroxyethyl methacrylate (HEMA) functionalized with N-methacryloyl-L-histidine methyl ester (MAH) were synthesized and used for the adsorption and separation of bovine IgG. Two series of cryogels functionalized with 5 and 10 mg of MAH as pseudobioaffinity ligand were prepared and characterized by swelling test, FTIR and SEM analysis. The adsorption efficiency of the bovine immunoglobulin into cryogels is discussed with respect to the following chromatographic parameters: pH, flow rate, initial IgG concentration, adsorption time and ionic strength. Our results show good adsorption of bovine immunoglobulin under mild separation conditions at pH 7.4. The maximum binding capacity was determined (32.4 mg/g of cryogel) and demonstrates the efficiency of the used cryogels. This efficacy is clearly seen upon increasing the maximum binding capacity from 23.2 mg (obtained with cryogels with 5 mg MAH) to 32.4 mg/g (for cryogel with 10 mg MAH ligand concentration). The purity of separated fractions was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Together our observations highlights poly (HEMA-MAH) as an efficient adsorbent for bovine immunoglobulins G separation.

Supermacroporous cryogels were synthesized and used for the purification of immunoglobulin G (IgG) from bovine serum. Imidazole functional groups were incorporated into basic polymeric backbone to gain pseudo-specificity to cryogels by using n-vinylimidazole as comonomer. The cryogels were prepared in aqueous solution of monomers partially frozen inside plastic syringe column by cryogelation. Poly(2-hydroxyethyl methacrylate-n-vinylimidazole) [poly(HEMA-VIM)] cryogels were prepared by polymerization of water-soluble functional monomer, n-vinylimidazole, with basic monomer HEMA. The characterization methods including swelling test, Fourier transform infrared spectroscopy (FTIR), elemental analysis, and scanning electron microscopy (SEM) were performed to evaluate physical and chemical properties of cryogels prepared. Bovine IgG adsorption on plain and composite cryogels was studied with respect to different parameters such as, pH, IgG concentration, flow rate, ionic strengths, and adsorption time. The best adsorption of bovine IgG was observed at pH 7.4 up to 21.1 mg per unit mass of poly(HEMA-VIM) cryogel. Elution of IgG adsorbed from the cryogels was easily achieved with 0.1 M acetate buffer containing 1 M NaCl at pH 4.0. In order to describe the adsorption process, we applied some equilibrium and kinetic adsorption models to the data. The results best fitted to Langmuir model showing monolayer protein adsorption and surface homogeneity of cryogel. Finally, we evaluated IgG purification from bovine serum under optimal condition determined. The purification efficiency and IgG purity were investigated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) study.

Immobilized metal ion affinity chromatography (IMAC) has been used for purification of proteins. IMAC introduces a new approach for selectively interacting biomolecules on the basis of their affinities for metal ions. The separation is based on different binding abilities of the proteins to the chelated metal ions on support. Here, N-methacryloyl-(L)-histidine methyl ester (MAH) is used as the metal-chelating ligand. Poly(hydroxyethyl methacrylate) Poly(HEMA) based membranes were prepared by photo-polymerization technique. Then, Zn(2+), Ni(2+), Co(2+), and Cu(2+) ions were chelated directly on the poly(HEMA-MAH) membranes for purification of immunoglobulin G (IgG) from human plasma.

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 2-3 μm, was estimated as 55.3 μmol MAH/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 24.7 mg insulin/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 (0.61 mg insulin/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.