Bovine Serum Albumin Immobilization Research Articles

Characterization of bovine serum albumin immobilization on surface modified glass slides in case of pyro-electrohydrodynamic spots

BackgroundPyro-electrohydrodynamic jetting (p-jet) has emerged recently as a promising technique for biosensing applications, through the concentration of highly diluted biomolecules in fluorescent spots at microscale. However, a great challenge still remains in optimizing the binding strategy for the sensing interface, enabling the detection of low abundance proteins through immunofluorescence protocols. Indeed, the surface of reaction can be functionalized with different chemical groups able to bind the target molecule with a strong interaction, prior to the p-jet spots decreasing the possibility to lose sensitivity after the common rinsing steps. ResultsHere, we characterize the immobilization of a model protein, specifically the bovine serum albumin (BSA), in the concentrated p-jet spots to demonstrate the reliability of the technique for highly sensitive immunodetection assays. We first performed spectroscopic measurements on BSA deposited through pipette spots at relatively high concentrations and we achieved a higher efficiency in case of the covalent bond by using the carbonate buffer and the epoxy-based slides. We then tested the covalent setting in case of the p-jet spots with highly diluted samples of pre-labelled BSA. A significant concentration-dependent behavior of the signal was obtained down to picogram levels. Finally, an immunofluorescent protocol was settled with the p-jet spots and a Limit of the Detection (LOD) of 0. 27 pg/mL was reached. SignificanceThe demonstration here that the p-jet spots are compatible with immunodetection procedures and provide a LOD down to 0.27 pg/mL, launches the p-jet technique towards the development in future of a point-of-care (POC) diagnostic tool. This would become a major force in analytical chemical laboratories. The identification of highly diluted biomarkers from peripheral body fluids would help clinicians performing early diagnosis, overcoming the limitations of the traditional immunochemistry tests, such as the enzyme-linked immunosorbent assay (ELISA).

Mass Spectrometric Identification of BSA Covalently Captured onto a Chip for Atomic Force Microscopy.

Mass spectrometry (MS) is one of the main techniques for protein identification. Herein, MS has been employed for the identification of bovine serum albumin (BSA), which was covalently immobilized on the surface of a mica chip intended for investigation by atomic force microscopy (AFM). For the immobilization, two different types of crosslinkers have been used: 4-benzoylbenzoic acid N-succinimidyl ester (SuccBB) and dithiobis(succinimidyl propionate) (DSP). According to the data obtained by using an AFM-based molecular detector, the SuccBB crosslinker was more efficient in BSA immobilization than the DSP. The type of crosslinker used for protein capturing has been found to affect the results of MS identification. The results obtained herein can be applied in the development of novel systems intended for the highly sensitive analysis of proteins with molecular detectors.

Total internal reflection ellipsometry for kinetics-based assessment of bovine serum albumin immobilization on ZnO nanowires

The properties of the ZnO-NWs were investigated by utilizing spectroscopic ellipsometry under TIRE configuration during real-time measurement of covalent BSA immobilization.

Metal coordination assisted thermo-sensitive magnetic imprinted microspheres for selective adsorption and efficient elution of proteins

Protein recognition and separation with high efficiency, low cost and facile construction has been hampered by the deficient development of fabrication methods. Toward this issue, we proposed a functionally integrated strategy by combining metal coordination, thermo-sensitive building blocks and hollow Fe3O4@mSiO2 microspheres (mSiO2 referred as mesoporous silica) with molecularly imprinted polymers (MIPs), to realize selective adsorption and effective desorption of the targeted protein bovine serum albumin (BSA). Herein, a series of metal ions (Mn2+, Fe3+, Ni2+, Cu2+, Zn2+) were selected to chelate onto the surface of amino groups modified substrates to investigate the metal coordination effect, wherein Ni2+ exhibited the optimal BSA immobilization ability. Beneficial from the sufficient templates coordinated by Ni2+, our MSS@Ni2+-MIPs (represented Ni2+-involved imprinted microspheres) with well-designed composite structure showed the best rebinding performance (Q = 182.18 mg/g, IF = 4.78) and rapid adsorption equilibrium kinetics (30 min) at 32 °C. Although template proteins were seized by the strong metal coordination, we obtained the satisfactory elution efficiency (61.97 %) at 20 °C assisted with the swollen thermo-sensitive components. Based on this facile, smart and versatile imprinting approach, the well-defined materials would hold great promising in bio-applications.

Evaluation of the interaction of levothyroxine with bovine serum albumin using spectroscopic and molecular docking studies

Bovine serum albumin (BSA) acts as a carrier for many endogenous and exogenous compounds, such as thyroid hormones or corresponding drugs. Binding of the hydrophilic levothyroxine drug (LT4) to BSA is of significant pharmacological importance. In this work, UV-vis measurements were used to determine the pH value at which LT4 interacts optimally with proteins. The binding mechanism and affinity of the interaction between LT4 and BSA were investigated using Fourier-transform infrared spectroscopy (FT-IR), fluorescence, fluorescence resonance energy transfer (FRET), Surface Plasmon Resonance (SPR), supplemented by molecular docking analysis. Fluorescence measurements revealed the quenching effect of LT4 on the BSA intrinsic fluorescence and LT4 binding with BSA is driven by a ground-state complex formation that may be accompanied by a nonradiative energy transfer process. The thermodynamic parameters correspond to an enthalpic process, driven mainly by hydrogen bonds and van der Waals forces. Using SPR, the adsorbed amount of biomolecules was calculated and the binding affinity of LT4 with confined-BSA was characterized, indicating that the BSA immobilization plays an important role in LT4 binding. Docking studies confirmed the formation of the LT4-BSA complex with LT4 bound to site I on the BSA structure mainly with amino acid residues Trp 213, Tyr 137, Tyr 147. The calculation of the apparent association constant confirms the result obtained in SPR. Communicated by Ramaswamy H. Sarma

A new tool for dengue virus diagnosis: Optimization and detection of anti-NS1 antibodies in serum samples by impedimetric transducers

An impedimetric immunosensor for the detection of anti-Non Structural 1 (anti-NS1) DENV, based on the specific non-Structural Protein 1 (rNS1) antibodies immobilized over poly(4-aminobenzoic acid)-modified screen-printed electrodes (SPE) was successfully developed for dengue diagnosis. The electropolymerization of 4-aminobenzoic acid in sulfuric acid 0.5 M solution was performed with 20 cycles at 100 mV.s−1 through cyclic voltammetry. Different rNS1 concentrations prepared in HBS-EP buffer solution, pH 7.4, were immobilized over the polymer-modified SPE surface via physical adsorption, and the electrode surface was blocked right after with bovine serum albumin (BSA). Electrochemical impedance spectroscopy (EIS) was chosen to detect the biomolecular interactions in the serum samples of immunized mice. Optimization studies regarding antigen immobilization time, BSA immobilization time, and antigen-antibody interaction time showed that 20.0, 5.0, and 30.0 min resulted in the best responses, respectively. Serum samples dilutions of 1:1280 showed a difference of 70% between the specific (Ab+) and non-specific (Ab−) samples. The proposed immunosensor showed good selectivity towards some interferents such as uric acid, glucose, water, HBS-EP buffer. Intra-day precision and repeatability were investigated, and a relative standard deviation (RSD) of 0.97 and 1.27% were found. Immunosensor stability was monitored for 45 days with storage at 4.0 °C with low response decrease. Validation analysis was conducted with human sera samples after confirmation with ELISA serological test. These results confirm that the developed immunosensor was able to detect anti-NS1 antibodies in real serum samples of patients infected with DENV, and this device can be a viable alternative for the diagnosis of this disease.

Separation and preconcentration of arsenite and other heavy metal ions using graphene oxide laminated with protein molecules

The graphene oxide surface was laminated with bovine serum albumin (BSA) followed by the directional flow through a membrane to prepare a free-standing PLGO (protein laminated GO) composite. BSA immobilization increased the interlayer spacing of GO and led to the formation of capillaries. The column packed with PLGO adsorbent permeated water faster as much as ca. 5 fold as compared to only GO packed column. The PLGO composite was used to develop a solid phase extraction method for the selective preconcentration of As(III) in the presence of As(V), prior to their determination. As(III) binding to sulfhydryl groups of BSA in PLGO plays a key role in the speciation. The coexisting heavy metal ions did not hinder the recovery of trace As(III). The method was advantageously employed for the preconcentration of As(III), Pb(II), Cd(II), Zn(II), Cu(II) and Ni(II) from water and food samples. A 3 mL of 1 M hydrochloric acid would be adequate for the complete desorption (recovery > 99%) of the adsorbed metal ions. The preconcentration limit achieved for As(III), Pb(II), Cd(II), Zn(II), Cu(II) and Ni(II) were 1.7, 2.0, 2.0, 2.0, 1.8 and 2.0 μg L−1 respectively, with an optimized sample flow rate of 10 mL min−1.

Label‐free Voltammetric Immunosensor for Prostate Specific Antigen Detection

AbstractThis work describes a sensitive voltammetric immunosensor for label‐free electroanalysis of the prostate specific antigen (PSA), the main biomarker of prostate cancer. A gold electrode was firstly modified with the optimum self‐assembled monolayer (SAM), 1,6‐hexanedithiol, followed by the subsequent adsorption of gold nanoparticles (AuNPs) and, then, the monoclonal antibody to recognize PSA was immobilized. The influence of the most significant experimental variables (SAM type and incubation time, AuNPs deposition, antibody concentration and bovine serum albumin immobilization) on the biosensor response was studied by microscopy and voltammetry techniques. The electroanalytical detection was based on the interaction between PSA antibody and PSA via square‐wave voltammetry using ferrocyanide/ferricyanide as electrochemical redox indicator. Using the proposed immunosensor, PSA was specifically detected within the linear range between 0.2 and 200 ng mL−1 with 0.01 ng mL−1 as limit of detection. The immunosensor allows accurate, reproducible and sensitive (22.7 % reduction mL ng−1) detection in a concentration range useful for clinical purposes.

Polysaccharide-based hydrogels for the immobilization and controlled release of bovine serum albumin

Arabic gum-based and chitosan-based hydrogels were synthesized through chemical crosslinking for the immobilization and controlled release of bovine serum albumin (BSA) and characterized by Fourier-transform infrared spectrometry, scanning electron microscopy and swelling assays. The degrees of swelling of the Arabic gum-based hydrogel were 13.22 and 22.95 g water per g dried hydrogel at pH 4.5 and 7.0, respectively, whereas the degrees of swelling of the chitosan-based hydrogel were 15.32 and 36.10 g water per g dried hydrogel, respectively. The water absorption mechanism in both hydrogels was non-Fickian, which involves diffusion through pores and macromolecular relaxation of the hydrophilic three-dimensional polymer network. BSA immobilization capacities of the Arabic gum-based and chitosan-based hydrogels after 240 min at pH 4.5 were 71.0 and 175.6 mg protein per g dried hydrogel, respectively. BSA immobilization capacities after 240 min at pH 7.0 were 62.5 and 154.2 mg protein per g dried hydrogel, respectively. The controlled release of BSA from the Arabic gum-based hydrogel was slightly more efficient than that of the chitosan-based hydrogel due to its more porous structure and weaker physiochemical interactions between the polymer network and protein molecule. Both hydrogels could be employed as carriers of proteins and as capsules for food supplements.

Kinetic and thermodynamic study of bovine serum albumin interaction with rifampicin using surface plasmon resonance and molecular docking methods.

The interaction of bovine serum albumin (BSA) with various drugs, such as antibiotics, due to the importance of BSA in drug delivery has attracted increasing research attention at present. Therefore, the aim of this study was investigation of BSA interaction with rifampicin using surface plasmon resonance (SPR) and molecular docking methods under the imitated physiological conditions ( pH = 7.4 ). BSA immobilization on carboxymethyl dextran hydrogel chip has been carried out after activation with N-hydroxysuccinimide/N-ethyl-N-(3-diethylaminopropyl) carbodiimide. The dose-response sensorgrams of BSA upon increasing concentration of refampicin were attained in SPR analysis. The high affinity of rifampicin to BSA was demonstrated by a low equilibrium constants ( K D ) value ( 3.46 × 10 ? 5 at 40°C). The process of kinetic values changing shows that affinity of BSA to rifampicin decreased with rising temperature. The positive value of both enthalpy change ( ? H ) and entropy change ( ? S ) showed that hydrophobic force plays major role in the BSA interaction with rifampicin. The positive value of ? G was indicative of nonspontaneous and enthalpy-driven binding process. In addition, according to the molecular docking study, hydrogen binding has some contributions in the interaction of rifampicin with BSA.

Stereoselective detection of amino acids with protein-modified single asymmetric nanopores

We demonstrate here facile approach to immobilize bovine serum albumin (BSA) protein molecules on the surface and inner walls of single asymmetric nanopores through mussel-inspired chemistry. Firstly, dopamine (DA) is allowed to self-polymerize under alkaline conditions, leading to the deposition of a thin and surface-adhered polydopamine (PDA) film on to the pore surface. Then, the BSA immobilization on PDA-coated pore surface is achieved via exploiting the amine groups on protein molecules and o-benzoquinone moieties through Michael addition reaction. The success of the chemical functionalization reactions is monitored through the changes in the corresponding current–voltage (I–V) curve. The immobilized BSA molecules serve as chiral receptor on the pore surface and have the ability to selectively bind l-enantiomer of tryptophan (l-Trp) amino acid. The specific BSA–tryptophan interactions inside the confined geometries lead to measurable changes in the electronic readout resulting from the modulation of ion current passing through the nanopore. The method permits l-Trp concentration detection in the range 100μM to 1.5mM. On the contrary, we did not notice any significant change in ion flux when the BSA-modified pore is exposed to phenylalanine (d-/l-Phe), d-tyrosine and d-tryptophan amino acids, separately. In this context, we believe that such nanoporous system can be extended for the detection and recognition of a variety of pharmaceutical molecules based on specific protein–drug interactions.

Capture and Release Erythrocyte from the Blood with Thermoresponsive and Core‐Sheath PCL/PNIPAAm Nanofibers

A newly developed nanofiber platform for nonadherent cell capture and release is demonstrated. The nanofiber platform is fabricated by single‐spinneret electrospinning of bovine serum albumin (BSA)‐conjugated poly(N‐isopropylacrylamide) (PNIPAAm) and polycaprolactone blends. The nanofibers possess core‐sheath structure with PNIPAAm as the sheath, which render the nanofibers switchable between hydrophilicity and hydrophobicity with the temperature change. As a result of BSA immobilization on the sheath part, the nanofibers resist platelet adhesion in the blood, facilitating the direct capture and isolation of red blood cells (RBCs) from the blood. Meanwhile, the captured RBCs are readily released from the nanofibers with the temperature stimuli. The capture and release efficiencies of up to 100% are achieved while maintaining cellular integrity and function. This work presents a promising platform to capture and release nonadherent cell effectively for subsequent molecular analysis and disease diagnosis.

Regioselective Localization and Tracking of Biomolecules on Single Gold Nanoparticles.

Selective localization of biomolecules at the hot spots of a plasmonic nanoparticle is an attractive strategy to exploit the light–matter interaction due to the high field concentration. Current approaches for hot spot targeting are time‐consuming and involve prior knowledge of the hot spots. Multiphoton plasmonic lithography is employed to rapidly immobilize bovine serum albumin (BSA) hydrogel at the hot spot tips of a single gold nanotriangle (AuNT). Regioselectivity and quantity control by manipulating the polarization and intensity of the incident laser are also established. Single AuNTs are tracked using dark‐field scattering spectroscopy and scanning electron microscopy to characterize the regioselective process. Fluorescence lifetime measurements further confirm BSA immobilization on the AuNTs. Here, the AuNT‐BSA hydrogel complexes, in conjunction with single‐particle optical monitoring, can act as a framework for understanding light–molecule interactions at the subnanoparticle level and has potential applications in biophotonics, nanomedicine, and life sciences.

BSA Adsorption and Immobilization onto Charged Monodisperse Polymer Nanoparticles

Controlling the behavior of adsorbed and immobilized proteins is essential for protein purification and a wide range of applications, including biosensors, biocatalysis and biomedical devices. In this study, monodisperse polymer particles were synthesized by soap free emulsion polymerization and the surface functional groups were directly introduced as a monomer or chemically modified with epoxy groups to enable protein immobilization through covalent bonding. Three kinds of surface charged particles having cationic, anionic, or both, groups were synthesized and characterized. Bovine serum albumin (BSA) was selected as a model protein to study the effect of pH of a buffer solution containing protein on the adsorption and immobilization amount. Protein adsorption was found to be strongly affected by pH and matching of the global protein and polymer particle charges, respectively. When pH was below the pI of either protein (pH 3.8), negatively charged polymer particles were found to adsorb a high amount of proteins. At maximum surface coverage of BSA on negatively charged polymer particles at pH 3.8 and after subsequent rinsing of the BSA-polymer particle complex with phosphate buffer (pH 7), ~50% of BSA was desorbed. Therefore, 50% of BSA was adsorbed by ionic interaction and the remaining fraction was immobilized covalently at pH 3.8. The remaining immobilized fraction was sufficient to completely shield the anionic charge of the polymer particles. Fluorescence spectroscopy suggests that at maximum immobilized amounts, the conformation of immobilized BSA appears to be the same as in aqueous solution.

Immobilisasi BSA pada Sensor QCM dengan Modifikasi Sifat Hidrofobik-Hidrofilik Permukaan Polistiren Menggunak

Research has been done to improve the optimization of the BSA ( Bovine Serum Albumin ) immobilization process on QCM sensor by modifying the nature of the hydrophobic-hydrophilic polystyrene surfaces using ultraviolet radiation (UV). The silver electrode of QCM sensor coated with a solution of polystyrene using spin coating technique. Sensor with polystyrene coating was heated using an oven until it reaches a temperature of 200 °C and then irradiated with ultraviolet light. A 30 µL of distilled water was dropped on the sensor surface and the formed contact angle was observed. The observation of the contact angle showed that there was a change in the nature hydrophobicity of sensor surface irradiated with UV. BSA immobilization was done by injecting a certain amount of BSA concentration in PBS solution on top of the sensor. We found that best surface for immobilization of BSA is one which is without UV irradiation. This means that the immobilization is mainly caused by hydrophobic force.

Functionalized polypropylene non-woven fabric membrane with bovine serum albumin and its hemocompatibility enhancement

Bovine serum albumin (BSA) was successfully immobilized onto polypropylene non-woven fabric (PPNWF) membranes using poly(acrylic acid) (PAA) as a spacer. Firstly, O2 plasma treatment and UV-irradiated technique were combined to graft PAA onto the membranes. BSA was then immobilized onto the PAA grafted surface through the coupling of amino groups of BSA to the carboxyl groups of PAA. The immobilization of PAA and BSA onto the membrane was confirmed by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), and water contact angle measurement. The water contact angle measurement results revealed that the membrane hydrophilicity improved after modification with PAA and BSA. After BSA immobilization, the amount of protein adsorption and the number of platelet adhesion on the modified membrane significantly decreased, which indicated that hemocompatibility had been considerably improved compared with neat and PAA grafted PPNWF. The whole blood clotting time measurement showed that the anticoagulant property of the modified membrane was also significantly enhanced.

Bioceramic Hydroxyapatite Granules for Purification of Biotechnological Products

Sorption experiments of bovine serum albumin (BSA) on hydroxyapatite (HAp) ceramic granules, prepared at three temperatures 900°C, 1000°C and 1150°C were performed at room temperature 18,6 °C and phosphate buffer, pH 5,83; 6.38 and 7,39. Thermal treatment contributed to the decrease of bovine serum albumin immobilization indicating that sorption process depended on HAp ceramics specific surface area and pH values of phosphate buffer solution. However, it was confirmed that granule size was also an important parameter for bovine serum albumin adsorption. As a result of these experiments, the most appropriate adsorption conditions and phosphate buffer pH values influence on to BSA sorption were analyzed.

Highly biocompatible multi-walled carbon nanotube–chitosan nanoparticle hybrids as protein carriers

Carbon nanotube (CNT)–organic polymer hybrids have important potential applications in the immobilization of therapeutic biomolecules. Recently developed CNT–organic polymer composites require the use of organic solvents for their preparation and have limited polymer functionalization. To address these limitations, multi-walled CNT (MWCNT)–chitosan nanoparticle (CS NP) hybrids have been synthesized in situ by an ionotropic gelation process, which is extremely mild and involves the mixture of two aqueous solutions at room temperature. The MWCNT–CS NP hybrids were characterized by atomic force microscopy and thermogravimetric analysis. Under optimal conditions the CS NP can be tethered to the MWCNT surface in high density and with relatively uniform coverage. The MWCNT–CS NP hybrids show good dispersibility and stability in aqueous solutions. In order to evaluate the potential utilization of the hybrids as protein carries the cytotoxicity to HeLa cells and protein immobilization (of bovine serum albumin (BSA), used here as a model) capacity of the hybrids were investigated in detail. The results demonstrate that the MWCNT–CS NP hybrids are biocompatible at concentrations up to 100μgmL−1 for 24h incubation. The MWCNT–CS NP hybrids improve the BSA immobilization efficiency 0.8 times and simultaneously decrease the cellular toxicity by about 50% compared with carboxylated MWCNT.

Poly(styrene-acrylamide-acrylic acid) copolymer fluorescent microspheres with improved hydrophilicity: preparation and influence on protein immobilization

Poly(styrene-acrylamide-acrylic acid) copolymer fluorescent microspheres (PSAAFMs) with improved surface hydrophilicity were synthesized through an improved soap-free emulsion copolymerization method, in which the proportion of acrylamide on the surface of the microspheres was increased. Azidocarbonyl groups, which can be rapidly coupled with proteins under mild conditions, were introduced onto the PSAAFMs using an azido reaction. The PSAAFMs were characterized using a fluorescence microscope, an ultraviolet/visible spectrometer, a Fourier transforms infrared spectrometer, a transmission electron microscope (TEM), a size analyzer, and a fluorescence spectrophotometer. Furthermore, covalent linking through the azidocarbonyl groups and physical nonspecific attachments of bovine serum albumin (BSA), trypsin, and human chorionic gonadotropin (HCG) onto the surface of the microspheres were also determined to evaluate the influence of improved surface hydrophilicity on nonspecific protein adsorption. Results from the TEM and size analyzer showed that the PSAAFMs maintained spherical shapes with an average diameter of 2.5 ± 0.22 µm. Fluorescence measurement indicated that the maximum emission wavelength underwent a slight blue shift from 514 to 512 nm. Environmental factors, such as pH value, imposed certain effects on fluorescence intensities. The linear relationship between fluorescence intensity and microspheres' concentration, which ranged from 1 × 10−3 to 10 × 10−3 g L−1, suggest their quantitative application. The significant decrease in the physical nonspecific adsorption of BSA, trypsin, and HCG in comparison with the microspheres without improved hydrophilicity suggest the increased amount of acrylamide on the surface of the microspheres. The protein covalent immobilization experiments revealed significant increases in BSA and HCG immobilization in comparison with the nonspecific physical attachment. The combination of high hydrophilicity and electrostatic repulsion could severely inhibit nonspecific protein attachment onto the surface of the microspheres.

Adsorption and bioactivity studies of albumin onto hydroxyapatite surface

Bovine serum albumin (BSA) may have an inhibitory or promoter effect on hydroxyapatite (HA) nucleation when apatite is precipitated in a medium containing the protein. In this study we evaluated the influence of BSA on the precipitation of calcium phosphate phases (CP) from simulated body fluid (SBF) when the protein was previously bounded to HA surface. The kinetics of BSA immobilization onto hydroxyapatite surface was performed in different buffers and protein concentrations in order to adjust experimental conditions in which BSA was tightly linked to HA surface for long periods in SBF solution. It was shown that for BSA concentration higher than 0.1mg/mL the adsorption to HA surface followed Langmuir–Freundlich mechanisms, which confirmed the existence of cooperative protein–protein interactions on HA surface. Fourier Transformed Infrared Attenuated Total Reflectance Microscopy (FTIRM-ATR) evidenced changes in BSA conformational state in favor of less-ordered structure. Analyses from high resolution grazing incident X-ray diffraction using synchrotron radiation (GIXRD), Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) showed that a poorly crystalline calcium phosphate was precipitated on the surface of HA discs coated with BSA, after the immersion in SBF for 4 days. The new bioactive layer had morphological characteristics similar to the one formed on the HA surface without protein. It was identified as a carbonated apatite with preferential crystal growth along apatite 002 direction. The GIXRD results also revealed that BSA layer bound to the surface inhibited the HA dissolution leading to a reduction on the formation of new calcium phosphate phase.