Bovine Serum Albumin Immobilization Research Articles

Preparation of magnetic iron/mesoporous silica composite spheres and their use in protein immobilization

The iron/silica magnetic composite spheres were prepared by electrochemical method and reduced in hydrogen atmosphere at different temperatures. The morphology and structure of the composite were characterized by SEM, TEM and XRD. The iron/silica microspheres exhibit essential ferromagnetic behavior characterized by magnetometry. After being coated with silica in sodium silicate solution by acidifying technology, the surface of these magnetic composite spheres is with amino-silane coupling agent for their attachment to affinity ligands. Bovine serum albumin (BSA) was covalently immobilized onto the amino-silane modified magnetic silica supports by the glutaraldehyde method. The influence of pH, ionic strength as well as the initial protein concentration on BSA immobilization was studied. The results show that such amino-silane modified magnetic composite spheres are the effective supports for bioseparation and the maximum BSA immobilization capacity (up to 87.4 mg/g) is obtained in 0.1 mol/L phosphate buffer at pH 5.0.

Synthesis and Characterization of Double-layer Quantum-Dots-Tagged Microspheres

Quantum-dots-tagged poly (styrene-acrylamide-acrylic acid) microspheres (QDsAAMs) were synthesized and modified with hydrazine hydrate through hydrazinolysis. Azidocarbonyl groups, which can be rapidly coupled with proteins under mild conditions, were introduced onto the surface of QDsAAM using azido reaction. Bovine serum albumin (BSA) was selected as model protein to be covalently immobilized on the azidocarbonyl QDsAAM. Instruments such as fluorescence microscope, optical microscope, confocal laser scanning microscope, UV-visible spectrometer, Fourier transform infrared spectrometer, size analyzer, and fluorescence spectrophotometer were used to characterize QDsAAM. Results showed that QDsAAM had a regular double-layer spherical shape and an average diameter of 11.2 microm. It also displayed high fluorescence intensity (lambda(ex)/lambda(em) = 250 nm/370 nm), which showed linearity with concentrations ranging from 3.0 x10(-3) to 90.0 x10(-3) g.L(-1). In addition, external factors such as pH and ionic strength exerted little influence on fluorescent characteristic. BSA immobilization indicated that QDsAAM with azidocarbonyl groups could be covalently coupled with BSA at the rate of 40 x10(-3) g/g (BSA/QDsAAM), while fluorescence linearity correlation was also found. This functional azidocarbonyl QDsAAM with sensitive fluorescence and active azidocarbonyl groups could be used as a promising fluorescent probe for quantitative detection, protein immobilization, and early rapid clinical diagnostics.

Carp vitellogenin detection by an optical waveguide lightmode spectroscopy biosensor

A label-free carp vitellogenin sensor has a strong potential for on-site monitoring on the possible contamination of edible fish with endocrine disruptors as a sum parameter in an inland carp farm. In this study, we performed a sensitive detection for carp vitellogenin with a direct-binding optical waveguide lightmode spectroscopy-based immunosensor. Carp vitellogenin bound over the sensor surface quite specifically, judging from the sensor responses according to stepwise antibody immobilization. This was also supported by a negligible sensor response found at bovine serum albumin immobilization. When plotted in double-logarithmic scale for carp vitellogenin concentrations of 0.00675–67.5 nM, a linear relationship was found between analyte concentration and sensor response, together with the limit of detection of 0.00675 nM. The reusability of the immunosensor after the regeneration with 10 mM HCl was reasonably good, as presumed from the coefficient of variability of 6.02% for nine repetitive measurements. The model sample prepared by spiking a purified carp vitellogenin into a 10-fold diluted vitellogenin-free carp serum in 9.45 nM showed the response ratio of 96.70% against 9.45 nM of the purified carp vitellogenin. When a female and male carp sera induced with 17β-estradiol injection were analyzed, biomarker induction was even identifiable at 2000-fold serum dilution.

Biocomposite of double-walled carbon nanotube-doped alginate gel for biomaterial immobilization

An efficient support for biomaterial immobilization, double-walled carbon nanotube-doped alginate gel (DWCNT–ALG) hybrid composite, was studied in this work. DWCNTs were cut by mixed acids to get a homogeneous dispersion in an aqueous solution. Fourier transform infrared spectroscopy (FTIR) was employed to analyze the structure of shortened DWCNTs. Bovine serum albumin (BSA), the model biomaterial, was immobilized in DWCNT–ALG composites. Mixture of DWCNTs and BSA were suspended in ALG homogenously and then added into a calcium chloride solution dropwise to form biocomposite beads. Transmission and scanning electron microscope (TEM and SEM) images confirmed the adsorption of BSA on DWCNTs and the uniform distribution of DWCNTs in ALG, respectively. Water loss and BSA leakage from the BSA–DWCNT–ALG biocomposites were reduced to 46.48% and 19.28%, respectively, compared with those from BSA–ALG biocomposites. These DWCNT–ALG biocomposites showed high efficiency in BSA immobilization.

Sulfamethazine detection with direct-binding optical waveguide lightmode spectroscopy-based immunosensor

A direct-binding optical waveguide lightmode spectroscopy-based immunosensor detecting sulfamethazine (SMZ) was prepared, followed by the measurement of its specificity and sensitivity. System construction was undertaken with a peristaltic pump, an injector and the main unit comprising a sensor holder, two signal-harvesting photodiodes, a beam mirror, shutter and He–Ne laser source emitting a monochrome light (λ=632.8nm), plus a PC. Antibody immobilization was performed in situ by covalent binding of an anti-SMZ antibody over the surface of a glutaraldehyde-activated 3-aminopropyltriethoxysilane-treated sensor chip. The reaction buffer for the system was 4mM Tris–HCl (pH 7.2) that showed a medium surface coverage and stable baseline. Sensor response was quite specific to antibody–antigen complexation, as judged from no sensor response caused by bovine serum albumin immobilization. The sensor responses according to SMZ concentrations from 10−8 to 10−2M increased linearly in a semi-logarithmic scale, with the limit of detection of 10−8M. The immunosensor was favorably reusable for SMZ screening.

BSA Adsorption on Hydroxyapatite after Thermal Treatment

Adsorption experiments of bovine serum albumin on hydroxyapatite previously annealed at temperatures up to 1100°C was performed at 37°C and phosphate buffer, pH 6.0. Kinetic process was very efficient and irreversible for low phosphate buffer concentration. Thermal treatment contributed to the decrease of bovine serum albumin immobilization indicating that sorption process depended on HA specific surface area and the number of surface active sites. However, it was verified that particle size was also an important parameter for bovine serum albumin immobilization.

Measurement of antibody binding to protein immobilized on gold nanoparticles by localized surface plasmon spectroscopy

Antibody binding to bovine serum albumin (BSA) and human serum albumin (HSA) immobilized onto gold nanoparticles was studied by means of localized surface plasmon resonance (LSPR) spectroscopy. Amine-modified glass was prepared by self-assembly of amine-terminated silane on substrate, and gold (Au) nanoparticles were deposited on the amine-modified glass substrate. Au nanoparticles deposited on the glass surface were functionalized by BSA and HSA. BSA immobilization was confirmed by LSPR spectroscopy in conjunction with surface-enhanced Raman scattering spectroscopy. Then, LSPR response attributable to the binding of anti-BSA and anti-HSA to BSA- and HSA-functionalized Au nanoparticles, respectively, was examined. Anti-HSA at levels larger than approximately 10 nM could be detected by HSA-immobilized chips with LSPR optical response, which was saturated at concentrations greater than approximately 650 nM of anti-HSA.

High-Capacity Binding of Proteins by Poly(Acrylic Acid) Brushes and Their Derivatives

Polymeric coatings with high protein-binding capacities are important for increasing the output of affinity-based protein purification and decreasing the detection limits of antibody microarrays. This report describes the use of thick poly(acrylic acid) (PAA) brushes to immobilize as much as 80 monolayers of protein. The brushes were prepared using a recently developed procedure that allows polymerization of 100-nm-thick poly(tert-butyl acrylate) films from a surface in just 5 min along with hydrolysis of these films to PAA in 15 min. Covalent binding of bovine serum albumin (BSA) to PAA brushes that were activated using standard coupling agents, however, resulted in immobilization of less than two monolayers of BSA because of competitive hydrolysis of the esters in the activated film. In contrast, derivatization of PAA with nitrilotriacetate (NTA)-Cu2+ complexes yielded films capable of binding many monolayers of protein via metal-ion affinity interactions. For example, derivatization of 55-nm-thick PAA films with NTA-Cu2+ allowed immobilization of about 15 monolayers (5.8 microg/cm2 or 58 nm) of BSA. The binding capacity was even higher for myoglobin (7.7 microg/cm2) and anti-IgG (9.6 microg/cm2). Remarkably, electrostatic adsorption of lysozyme in 55-nm-thick, underivatized PAA resulted in as much as 80 monolayers (16.2 microg/cm2 or 162 nm) of adsorbed protein. Polymer synthesis, derivatization, and swelling, as well as BSA immobilization kinetics and thermodynamics were characterized using reflectance FT-IR spectroscopy, ellipsometry, and protein assays.

Nanotube-doped alginate gel as a novel carrier for BSA immobilization

In this paper different dopants, including carbon nanotubes (CNTs), silica nanotubes (SiNTs), graphite and silica gel, were incorporated into alginate (ALG) gel to form nanotube/alginate or nanoparticle/alginate composites used for bovine serum albumin (BSA) immobilization carrier. During encapsulation, first BSA was adsorbed on the dopants, and then BSA and dopants were suspended in alginate solution, followed by being added dropwise into the CaCl2 solution to form the biocomposites. The BSA leakage decreased significantly in these biocomposites as the following order: BSA-ALG-SiNT > BSA-ALG-CNT > BSA-ALG-SiO2 > BSA-ALG-graphite, which was mainly due to firstly the protein molecule adsorption on the dopants which could not be washed out easily and secondly, during the biocomposites formation, water loss in BSA-ALG-dopant biocomposites became less than that in BSA-ALG biocomposite. In addition, the mechanical properties of these biocomposites were remarkably reinforced in the same order as BSA leakage decrease.

Synthesis and protein immobilization of monodisperse magnetic spheres with multifunctional groups

Monodisperse magnetic spheres with multifunctional groups were synthesized by spraying suspension polymerization. The effects of stabilizer type and its concentration on the monodispersity of magnetic spheres were investigated. The results showed that polyvinyl alcohol (PVA) was satisfactory as a stabilizer in this spraying suspension polymerization, and the magnetic spheres showed good monodispersity when the concentration of PVA was increased to above 20 wt%. It was also found that the monodispersity of magnetic spheres could be improved with increase of the amount of initiator. Scanning electron microscopy and vibrating sample magnetometer verified that the magnetic spheres obtained were monodisperse with small size (about 9.8 μm) and displayed superparamagnetic characteristics. Data of Fourier transform infrared (FT-IR) spectroscopy and surface chemical reaction method indicated that the extensive epoxy groups existed on the surface of magnetic spheres. Bovine serum albumin (BSA) was covalently immobilized onto the glutaraldehyde treated and untreated magnetic spheres, respectively. The results showed that the maximum BSA immobilization capacity onto the magnetic spheres was about 57 mg/g.

Synthesis and characterization of surface-hyperbranched magnetite nanoparticle for bovine serum albumin immobilization

A hyperbranched polyamidoamine polymer was synthesized on the surface of magnetite nanoparticles to enhance bovine serum albumin (BSA) immobilization efficiency. The amount of immobilized bovine serum albumin (BSA) on the surface-hyperbranched magnetite nanoparticle was up to 2.5 times as much as that of magnetite nanoparticle modified with only amino silane.

BSA Immobilization on Amine-Functionalized Superparamagnetic Iron Oxide Nanoparticles

Immobilization of bovine serum albumin (BSA) on surface-modified superparamagnetic iron oxide nanoparticles (SPION) has been performed by two different double-step immobilization approaches. The first approach consists of preparation of SPION by controlled chemical coprecipitation in the presence of BSA solution, whereas the second approach includes preliminary surface modification of SPION with an amine group using a coupling agent of 3-aminepropyltrimethoxysilane (APTMS). Both procedures are followed by 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide hydrochloride (EDC) activation with sequential immobilization of the layer of BSA. Additionally, an attempt to modify the surface of SPION with amine and carboxylic groups is undertaken by using l-aspartic acid (LAA). TEM shows that the particle size varies in the range 10−15 nm and does not change significantly after the coating process. The presence of BSA and amine groups on the surface of SPION is confirmed by FT-IR. Magnetic properties are investigated by VSM and results indicate that the superparamagnetic properties are retained for BSA-coated SPION while reducing the value of saturation magnetization (Ms). The binding capacity is estimated from thermogravimetric and chemical analyses. APTMS-coated SPION show higher BSA binding capacity compared to that of coprecipitated SPION in the presence of BSA. In vitro tests have been performed after the functionalization of SPION with LAA and BSA. Human dermal fibroblasts are incubated with the surface-modified SPION for 6 and 24 h to observe cell behavior, morphology, cytoskeletal organization, and interactions between cell and SPION. BSA-coated SPION incubated with cells demonstrated a cell response similar to that of control cells, with no adverse cell damage and no endocytosis, whereas LAA-coated SPION show partial endocytosis without cytoskeletal disorganization.

Synthesis of amino-silane modified superparamagnetic silica supports and their use for protein immobilization

A novel method to synthesize amino-silane modified magnetic silica supports was reported for affinity separation. The magnetite (Fe 3O 4) nanoparticles were prepared by the coprecipitation of ferrous and ferric salts with NH 4OH, and then their surface was coated with silica in sodium silicate solution by acidifying technology to form well-dispersed magnetic silica nanospheres. These magnetic silica nanospheres are of about 50–80 nm in diameter and exhibit superparamagnetic characteristics. The surface of these nanospheres was with amino-silane coupling agent for their attachment to affinity ligands. In this work, bovine serum albumin (BSA) was covalently immobilized onto the amino-silane modified magnetic silica supports by the glutaraldehyde method. The influence of pH, ionic strength as well as the initial protein concentration on BSA immobilization has been studied. The result shows that such amino-silane modified magnetic silica is a well-dispersed and effective superparamagnetic support for bioseparation and the maximum BSA immobilization capacity (up to 86 mg/g) was obtained in 0.1 M phosphate buffer at pH 5.0.