RESUMEN
Polyethyleneimine (PEI) coated-silica nanoparticles were prepared by the Stöber method. The formation and the structure of the nanoparticles were characterized by ATR-FT-IR spectroscopy and transmission electron microscopy (TEM). TEM images of the silica and PEI-coated nanoparticles revealed that they were well dispersed and that there was no agglomeration. The acetylcholineesterase enzyme was immobilized onto these nanoparticles. The effects of pH and temperature on the storage stability of the free and immobilized enzyme were investigated. The optimum pHs for free and immobilized enzymes were determined as 7.0 and 8.0, respectively. The optimum temperatures for free and immobilized enzymes were found to be 30.0 and 35.0°C, respectively. The maximum reaction rate (Vmax) and the Michaelis-Menten constant (Km) were investigated for the free and immobilized enzyme. The storage stability of acetylcholinesterase was increased when immobilized onto the novel PEI-coated silica nanoparticles. The reuse numbers of immobilized enzyme were also studied. These hybrid nanoparticles are desirable as carriers for biomedical applications.
Asunto(s)
Acetilcolinesterasa/química , Enzimas Inmovilizadas/química , Nanopartículas/química , Polietileneimina/química , Dióxido de Silicio/química , Acetilcolinesterasa/metabolismo , Estabilidad de Enzimas , Enzimas Inmovilizadas/metabolismo , Concentración de Iones de Hidrógeno , TemperaturaRESUMEN
Modifying the surfaces of magnetic nanoparticles (MNPs) by the covalent attachment of biomolecules will enable their application as media for magnetically-assisted bioseparations. In this paper, we reported both the activity and specific binding capacity of ferritin antibodies on core-shell MNPs. The antibodies were covalently attached on silica-, silver- and polydopamine-coated MNPs by different methods. Anti-ferritin was bound onto the silica- or silver-coated MNPs by conventional methods using 3-aminopropyltriethoxysilane (APTES) or 11-mercaptoundecanoic acid (MUA), which was followed by activation of carboxyl groups by EDC/NHS. However with anti-ferritin immobilized onto the Fe3O4 nanoparticles modified with polydopamine, an in situ coating formed through the adhesive proteins. In addition, a great deal of anti-ferritin biomolecules covalently attached onto the MNPs. According to our results, the amounts of bound anti-ferritin onto the silica-, silver- and PDA-coated MNPs were 70, 75 and 95 µg anti-ferritin per mg MNP, respectively. In the experiments, polydopamine (PDA)-coated MNPs showed faster adsorption, more significant selectivity and a larger binding capacity than the others. Also, the equilibrium dissociation constants of the antigen-antibody complexes were determined on the anti-ferritin-immobilized MNPs. Silica-, PDA- and silver-coated MNPs had Kd values of 5.45 × 10(-7), 2.12 × 10(-7) and 3.91 × 10(-8) mol L(-1), respectively. Based on these results, the affinity of the anti-ferritin for ferritin on the PDA-coated MNPs was approximately 10-fold higher than that on the silica- and silver-coated MNPs. In addition, among the anti-ferritin-immobilized silica-, silver- and PDA-coated MNPs, the PDA-coated MNPs showed the highest antigen selectivity values. As a result, anti-ferritin-immobilized PDA-coated MNPs represented a higher activity and stronger affinity for the specific antigen than the others.
Asunto(s)
Ferritinas/análisis , Ferritinas/metabolismo , Nanopartículas de Magnetita/química , Animales , Caballos , Humanos , Ratones , Unión Proteica/fisiología , ConejosRESUMEN
Core-shell magnetic nanoparticles (MNPs) offer tremendous opportunities in a large range of applications in biomedicine due to their superior magnetic properties, biocompatibility and suitability for modification. In most cases, these characteristic features are determined by their shell chemistry and morphology. Herein, we demonstrate a comparative study of silica and polydopamine (PDOP) coating onto MNP surfaces based on synthesis, characterization and usage in a bio-separation platform. It was found that monodispersed MNPs may be easily obtained on silica coating of varying shell thickness, whereas a continuous PDOP layer observed around the MNPs prevents the formation of the dispersed form. On the other hand, PDOP coated MNPs exhibited better superparamagnetic behavior and biological modification ability compared to the silica coated form.
Asunto(s)
Inmunoglobulina G/aislamiento & purificación , Indoles/química , Nanopartículas de Magnetita/química , Polímeros/química , Dióxido de Silicio/química , Anticuerpos Antiidiotipos , Inmunoglobulina G/inmunología , MagnetismoRESUMEN
We have developed a strategy to immobilize ß-galactosidase as a model enzyme by using polymeric supports having Schiff bases, which were prepared from (aminomethyl)polystyrene and 2-phenlyindole-3-carboxaldehyde by condensation. ß-galactosidase was immobilized onto the new polymer supports via covalent bonds. The influence of temperature, pH, reusability, and storage capacity on the free and immobilized ß-galactosidase was investigated. Our results indicate that the (aminomethyl)polystyrene with Schiff bases is most suitable for the immobilization of ß-galactosidase. These kinds of new supports can be used for the immobilization of ß-galactosidase due to their strong storage capacity and reusability.
Asunto(s)
Enzimas Inmovilizadas/química , Polímeros/química , Bases de Schiff/química , beta-Galactosidasa/química , Aldehídos/química , Estabilidad de Enzimas , Enzimas Inmovilizadas/metabolismo , Escherichia coli/enzimología , Concentración de Iones de Hidrógeno , Cinética , Poliestirenos/química , Temperatura , beta-Galactosidasa/metabolismoRESUMEN
A new method for immobilization of acetylcholinesterase (AChE) to alginate gel beads by activating the carbonyl groups of alginate using carbodiimide coupling agent has been successfully developed. Maximum reaction rate (V (max)) and Michaelis-Menten constant (K (m)) were determined for the free and binary immobilized enzyme. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized AChE were also investigated. For the free and binary immobilized enzyme on the Ca-alginate gel beads, optimum pH values were found to be 7 and 8, respectively. Optimum temperatures for the free and immobilized enzyme were observed to be 30 and 35 degrees C, respectively. Upon 60 days of storage the preserved activity of free and immobilized enzyme were found as 4 and 68%, respectively. In addition, reuse number, and thermal stability of the free AChE were increased by as a result of binary immobilization.
Asunto(s)
Acetilcolinesterasa/química , Alginatos/química , Técnicas Biosensibles/métodos , Materiales Biocompatibles Revestidos/química , Activación Enzimática , Estabilidad de Enzimas , Enzimas Inmovilizadas/química , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Concentración de Iones de Hidrógeno , Ensayo de Materiales , Microesferas , TemperaturaRESUMEN
Jack bean urease (urea aminohydrolase, E.C. 3.5.1.5) was entrapped into chitosan-alginate polyelectrolyte complexes (C-A PEC) and poly(acrylamide-co-acrylic acid)/kappa-carrageenan (P(AAm-co-AA)/carrageenan) hydrogels for the potential use in immobilization of urease, not previously reported. The effects of pH, temperature, storage stability, reuse number, and thermal stability on the free and immobilized urease were examined. For the free and immobilized urease into C-A PEC and P(AAm-co-AA)/carrageenan, the optimum pH was found to be 7.5 and 8, respectively. The optimum temperature of the free and immobilized enzymes was also observed to be 55 and 60 degrees C, respectively. Michaelis-Menten constant (K(m)) values for both immobilized urease were also observed smaller than free enzyme. The storage stability values of immobilized enzyme systems were observed as 48 and 70%, respectively, after 70 days. In addition to this, it was observed that, after 20th use in 5 days, the retained activities for immobilized enzyme into C-A PEC and P(AAm-co-AA)/carrageenan matrixes were found as 55 and 89%, respectively. Thermal stability of the free urease was also increased by a result of immobilization.
Asunto(s)
Acrilamidas/química , Alginatos/química , Carragenina/química , Quitosano/química , Fabaceae/enzimología , Ureasa/química , Adsorción , Estabilidad de Enzimas , Enzimas Inmovilizadas/química , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Unión ProteicaRESUMEN
In this study, a new matrix for immobilization of acetylcholinesterase was investigated by using alginate and kappa-carrageenan. The effects of pH, temperature, storage and thermal stability on the free and immobilized acetylcholinesterase activity were examined. Maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) was also investigated for free and immobilized enzymes. For free and immobilized enzymes into Ca-alginate and alginate/kappa-carrageenan polymer blends, optimum pH and temperature was found to be 7 and 30 degrees C, respectively. For free enzyme, maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) values were found to be 6.35 mM and 50 mM min(-1), respectively, the same values for immobilized enzymes were determined as 8.68, 12.7 mM and 39.7, 52.9 mM min(-1), respectively. Storage and thermal stability of acetylcholinesterase was increased by as a result of immobilization.
Asunto(s)
Acetilcolinesterasa/química , Materiales Biocompatibles/química , Enzimas Inmovilizadas/química , Alginatos/química , Animales , Carragenina/química , Electrophorus , Estabilidad de Enzimas , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Calor , Concentración de Iones de Hidrógeno , Cinética , Polímeros/química , Desnaturalización Proteica , Manejo de Especímenes , Temperatura , Factores de TiempoRESUMEN
The use of the immobilized and the stable enzymes has immense potential in the enzymatic analysis of clinical, industrial and environmental samples. However, their widespread uses are limited due to the high cost of their production. In this study, binary immobilization of tyrosinase by using Ca-alginate and poly(acrylamide-co-acrylic acid) [P(AAm-co-AA)] was investigated. Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for the free and binary immobilized enzymes. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized tyrosinase were also examined. For the free and binary immobilized enzymes on Ca-alginate and P(AAm-co-AA), optimum pH was found to be 7 and 5, respectively. Optimum temperature of the free and immobilized enzymes was observed to be 30 and 35 degrees C, respectively. Reuse number, storage and thermal stability of the free tyrosinase were increased by a result of binary immobilization.