Covalent Binding Method Research Articles

α‐Amylase immobilization on functionalized nano CaCO3 by covalent attachment

AbstractIn this study, α‐amylase was immobilized on glutaraldehyde activated silanized calcium carbonate nanoparticles by a using covalent binding method. The surface modified nano calcium carbonate (CaCO3) were characterized using FTIR and SEM. Immobilization yield was found as 199.43 mg/g of calcium carbonate nanoparticles. The maximum activity was observed at pH 6.5. The immobilized enzyme had a higher activity at elevated temperature (50–90°C) than the free one. Reuse studies demonstrated that the immobilized enzyme could reuse 25 times while retaining 18.2% of its activity. Free enzyme lost its activity completely within 15 days. Vmax values for the free and immobilized enzymes were calculated as 10 and 0.35 mg/mL/min, respectively.

Immobilization of Candida rugosa lipase on glass beads for enantioselective hydrolysis of racemic Naproxen methyl ester

Candida rugosa lipase (CRL) was immobilized on glutaraldehyde-activated aminopropyl glass beads by using covalent binding method or sol-gel encapsulation procedure and improved considerably by fluoride-catalyzed hydrolysis of mixtures of RSi(OCH3)3 and Si(OCH3)4. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e. the hydrolysis of p-nitrophenylpalmitate (p-NPP). It has been observed that the percent activity yield of the encapsulated lipase was 166.9, which is 5.5 times higher than that of the covalently immobilized lipase. The enantioselective hydrolysis of racemic Naproxen methyl ester by immobilized lipase was studied in aqueous buffer solution/isooctane reaction system and it was noticed that particularly, the glass beads based encapsulated lipases had higher conversion and enantioselectivity compared to covalently immobilized lipase. In short, the study confirms an excellent enantioselectivity (E>400) for the encapsulated lipase with an ee value of 98% for S-Naproxen.

Collagen-functionalised electrospun polymer fibers for bioengineering applications

Polymer electrospun fibers are gaining increasing importance in nanobiotechnology, due to their intrinsic three-dimensional topography and biochemical flexibility. Here we present an in-depth study of protein functionalisation for polymethylmethacrylate fibers. We compare different coating approaches for type I collagen, including physisorption and covalent binding methods relying on functional linkers. The biofunctionalised fibers are investigated by scanning electron and confocal laser scanning microscopy, wettability measurements, Fourier-transform infrared spectroscopy, and protein quantification assays. We demonstrate that the largest amount of proteins adsorbed on fibers does not determine the best performance in terms of cell attachment and proliferation in vitro, which is instead related to the type of linking and the relevant role played by adsorption of serum biomolecules on the three-dimensional nanostructures. This study is relevant for designing and engineering novel biomaterials and scaffold architectures based on electrospun nanofibers.

Urea degradation kinetics in model wine solutions by acid urease immobilised onto chitosan-derivative beads of different sizes

In this work, a purified acid urease preparation was covalently immobilised onto porous chitosan beads of different size. The covalent binding method was found to be more efficient than the adsorption cross-linkage one whatever the glutaraldehyde-to-chitosan bead ratio ( Y GA/CHI) used. At the optimal Y GA/CHI ratio of 0.625 g g −1 , the specific activity ( A Bi) of the biocatalysts decreased from circa 300 to 70 IU g −1 wet support, as the bead average diameter ( d P) increased from 0.14 to 2.2 mm. Generally, A Bi reduced less than 5% after preservation in the wet form at 4 °C for 150–170 days. Only the biocatalyst prepared using the Chitopearl BCW-3001 lost about 40% of its initial activity. The kinetics of urea degradation in a model wine solution using these biocatalysts was of the pseudo-first order with respect to the urea concentration in the liquid bulk, the apparent pseudo-first order kinetic rate constant ( k Ii) ranging from about two thirds to one fifth of that ( k If) pertaining to free acid urease. In the operating conditions tested, the reaction kinetics was estimated as unaffected by the contribution of the external film and intraparticle diffusion mass-transfer resistances. When the model wine solution was enriched with the high-inhibitory tannins extracted from grape seeds, at the maximum level tested (374 ± 2 g GAE m −3) k Ii reduced to no more than (58 ± 9)% of k If, this proving quite a higher protective action against such compounds for the chitosan-based biocatalysts towards free or Eupergit ® C 250 L-immobilised acid urease.

Analysis of interaction between liposome membranes induced by stress condition: Utilization of liposomes immobilized on indium tin oxide electrode

NBD-cholesterol (NBD-Ch)-modified liposome was immobilized on indium tin oxide (ITO) electrode via the covalent binding method. The transfer of NBD-Ch between the immobilized liposomes and the target liposomes was observed by using a fluorescent microscope. The addition of liposome suspension co-incubated with alpha-chymotrypsin or stimuli-responsive polymer to the surface of the above ITO electrode, enhanced the liposome-liposome interaction, resulting in the promotion of NBD-Ch transfer. The apparent transfer rate constant of NBD-Ch was found to be correlated with the index for the liposome-liposome interaction evaluated by an immobilized liposome chromatography. This suggests that the present method using the liposome-immobilized ITO electrode was effective to evaluate the liposome-liposome interaction induced by the protein or the stimuli-responsive polymer under stress conditions.

Surface modification of Mitoxantrone-loaded PLGA nanospheres with chitosan

The purpose of this research was to develop polylactic-co-glycolic acid (PLGA) nanospheres surface modified with chitosan (CS). Mitoxantrone- (MTO-) loaded PLGA nanospheres were prepared by a solvent evaporation technique. The PLGA nanospheres surface was modified with CS by two strategies (adsorption and covalent binding). PLGA nanospheres of 248.4 ± 21.0 nm in diameter characterized by the laser light scattering technique, scanning electron microscopy (SEM) are spherical and its drug encapsulation efficiency is 84.1 ± 3.4%. Zeta potential of unmodified nanospheres was measured to be negative −21.21 ± 2.13 mV. The positive zeta potential of modified nanospheres reveals the presence of CS on the surface of the modified nanospheres. Modified nanospheres were characterized for surface chemistry by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT-IR). FT-IR spectra exhibited peaks at 3420 cm −1 and 1570 cm −1, XPS spectra shows the N 1s (atomic orbital 1s of nitrogen) region of the surface of the nanospheres, corresponding to the primary amide of CS. In vitro drug release demonstrated that CS-modified nanospheres have many advantages such as prolonged drug release property and decreased the burst release over the unmodified nanospheres, and the modified nanospheres by covalent binding method could achieve the release kinetics of a relatively constant release. These data demonstrate high potential of CS-modified PLGA nanospheres for the anticancer drug carrier.

Immobilization of intact liposomes on solid surfaces: A quartz crystal microbalance study

Immobilization of model cell membranes (liposomes) on sensing devices is useful for the detection, evaluation, and analysis of interaction between cell membranes and proteins or other biological materials. The method to immobilize the liposomes on the solid surface was investigated by using the quartz crystal microbalance (QCM) method, focusing on the density of immobilized liposomes and their stability. The different liposomes were applied onto the planar surface of the QCM electrode by varying the immobilization method. The results showed that the immobilized liposomes depended not only on the immobilization method but also on the properties of the liposomes. The liposome with low membrane fluidity likely showed intact immobilization on a solid surface. An electrostatic interaction also affected the amount of immobilized liposomes on the functionalized quartz crystal. By using the amino-coupling method, intact liposomes could be immobilized on a solid surface and immobilized liposomes could stabilize for 10 h with frequency changes less than 5%.

Microfluidic conductimetric bioreactor

A microfluidic conductimetric bioreactor has been developed. Enzyme was immobilized in the microfluidic channel on poly-dimethylsiloxane (PDMS) surface via covalent binding method. The detection unit consisted of two gold electrodes and a laboratory-built conductimetric transducer to monitor the increase in the conductivity of the solution due to the change of the charges generated by the enzyme-substrate catalytic reaction. Urea–urease was used as a representative analyte-enzyme system. Under optimum conditions urea could be determined with a detection limit of 0.09mM and linearity in the range of 0.1–10mM (r=0.9944). The immobilized urease on the microchannel chip provided good stability (>30 days of operation time) and good repeatability with an R.S.D. lower than 2.3%. Good agreement was obtained when urea concentrations of human serum samples determined by the microfluidic flow injection conductimetric bioreactor system were compared to those obtained using the Berthelot reaction (P<0.05). After prolong use the immobilized enzyme could be removed from the PDMS microchannel chip enabling new active enzyme to be immobilized and the chip to be reused.

Immobilisation of horseradish peroxidase onto monodisperse poly(glycidyl methacrylate) microspheres

The aim of this study was to immobilise horseradish peroxidase (HRP) onto poly(glycidyl methacrylate) (PGMA) microspheres. In this study, PGMA particles were synthesised by dispersion polymerization from the glycidyl methacrylate (GMA) monomer. The monomer conversion value was determined to be 89% by weight. The first step comprised opening of the epoxide rings and formation of amine groups on the particle surfaces. The characterised particles were used as micro-carriers for the immobilisation of HRP. Glutaraldehyde, as bifunctional reactive, has been used as the spacer arm in the second step of the covalent binding method. The optimum immobilisation conditions found are as follows: immobilisation time 10 min, pH 7.2, temperature 25°C, initial enzyme concentration 0.05 mg/ml. Kinetic parameters of native and immobilised enzyme, such as K m and V max values, have been calculated 2.33 mmol/l and 0.670 mmol/l per s for the native enzyme and for the immobilised enzyme 69.13 mmol/l and 0.175 mmol/l per s, respectively. The HRP immobilised microparticules were used to purify a textile bleaching solution in which excess H2O2 remains.

3-Mercaptopropionic Acid modified Porous Silicon Substrate used in Hyperammonemia

ABSTRACTAmperometric urea sensor is more suitable than optical and potentiometric urea sensor to diagnose hyperammonemia. However, because sensitivity in low concentration decreases remarkably, despite amperometric urea sensor has been studied for a long time it has not been applied for clinical diagnosis. In this paper, a new structure for an amperometric urea sensor was fabricated by MEMS, electrochemical etching, and electrostatic covalent binding techniques. Until now most amperometric urea sensors have had a membrane fixed on top of the transducer. That method often leads to malfunction of the sensor, arising from problems such as inadequate membrane adhesion, insufficient mechanical stability, and low sensitivity. To solve these kinds of problems, urease (Urs) was immobilized by electrostatic covalent binding method on the porous silicon (PSi) substrate coated self-assembled monolayer (SAM). Electrostatic covalent binding method was used to keep anisotropic orientation of urease on SAM.

Novel phosphate–phosphonate hybrid nanomaterials applied to biology

A new process for preparing oligonucleotide arrays is described that uses surface grafting chemistry which is fundamentally different from the electrostatic adsorption and organic covalent binding methods normally employed. Solid supports are modified with a mixed organic/inorganic zirconium phosphonate monolayer film providing a stable, well-defined interface. Oligonucleotide probes terminated with phosphate are spotted directly to the zirconated surface forming a covalent linkage. Specific binding of terminal phosphate groups with minimal binding of the internal phosphate diesters has been demonstrated. On the other hand, the reaction of a bisphosphonate bone resorption inhibitor (Zoledronate) with calcium deficient apatites (CDAs) was studied as a potential route to local drug delivery systems active against bone resorption disorders. A simple mathematical model of the Zoledronate/CDA interaction was designed that correctly described the adsorption of Zoledronate onto CDAs. The resulting Zoledronate-loaded materials were found to release the drug in different phosphate-containing media, with a satisfactory agreement between experimental data and the values predicted from the model.

Metal Phosphonates Applied to Biotechnologies: A Novel Approach to Oligonucleotide Microarrays

A new process for preparing oligonucleotide arrays is described that uses surface grafting chemistry which is fundamentally different from the electrostatic adsorption and organic covalent binding methods normally employed. Solid supports are modified with a mixed organic/inorganic zirconium phosphonate monolayer film providing a stable, well-defined interface. Oligonucleotide probes terminated with phosphate are spotted directly on to the zirconated surface forming a covalent linkage. Specific binding of terminal phosphate groups with minimal binding of the internal phosphate diesters has been demonstrated. The mixed organic/inorganic thin films have also been extended for use arraying DNA duplex probes, and therefore represent a viable general approach to DNA-based bioarrays. Ideas for interfacing mixed organic/inorganic interfaces to other bioapplications are also discussed.

Synthesis of diagnostic test kits for syphilis with polymeric particles

This investigation aimed to derive alternative guidelines for syphilis. In the first step of this study monodisperse PGMA particles were synthesised by dispersion polymerization of the GMA monomer by using AIBN as initiator and PAA as stabilizator. A series of characterization experiments were done. In the second step of the study the characterized particles were used as microcarriers for the immobilization of the natural antigen for the purpose of diagnosing syphilis. The covalent binding method was used and GA was utilized as a spacer arm. The optimum immobilization conditions for the binding of natural antigen onto the particles were determined. In the third and the last step of the study the agglutination tests were realized with in vitro experiments by using both serum samples taken from syphilis patients and positive controls. After these experiments the kits chosen among the ones showing high performance were compared with the commercial RPR kit being used widely by reacting with serially diluted positive controls and patient serums. After this comparison the performances of the synthesized kits were found as satisfactory as the commercial kits.

Hydrolysis of starch particles using immobilized barley α-amylase

Barley α-amylase has been immobilized on silica particles with diameters between 0.5 and 10 μm using a covalent binding method. Immobilization procedures were adjusted to optimize enzyme activity. The effects of product inhibition, thermal stability and operational stability have been determined. The feasibility of using the immobilized enzyme to hydrolyze wheat starch particles at temperatures below the gelatinization temperature (<55 °C) was proven. The optimal conditions for the hydrolysis were found to be: pH 4.5, 40 °C, calcium ion concentration 0.002 M and immobilized enzyme loading of 30 mg/ml. At these conditions, the immobilized enzyme was able to hydrolyze wheat starch particles at concentrations as high as 100 mg/ml with a final conversion of 90% after 24 h of operation. Maltose and glucose were found to inhibit the immobilized enzyme in a similar manner as reported previously using soluble enzyme. Although the thermostability of the immobilized enzyme was superior to the soluble enzyme, the immobilized enzyme degraded at the same rate as the soluble enzyme during cold wheat starch hydrolysis (operational stability unchanged). Model equations are presented for product inhibition, hydrolysis kinetics and enzyme degradation. Using best-fit parameters, the equations are shown to fit the experimental data well.

LACCASE-BASED SCREEN PRINTED ELECTRODE FOR AMPEROMETRIC DETECTION OF PHENOLIC COMPOUNDS

ABSTRACT A laccase-based screen-printed electrode for monitoring phenolic compounds in environmental samples is described. The selectivity and sensitivity of laccase-catalyzed phenol oxidation were combined with the advantages of the screen-printing microfabrication technology. Laccase was immobilized over the screen-printed carbon surface by simultaneous cross-linking and covalent binding methods. Variables relevant to laccase immobilization on the screen-printed electrode surface were examined and optimized. This sensor allowed the convenient quantification of guaiacol and oguaiacol down to 50 and 30 nmol L−1, respectively, and selective measurements in untreated river and groundwater samples. These, and their attractive performance characteristics, are described.

Covalent immobilization of protein monolayers for biosensor applications

Various sensor technologies require a monolayer of tightly bound proteins. Five methods for protein immobilization were evaluated, comprising two methods of linker attachment to a silicon nitride surface (CNBr, silanization) and three methods of linker attachment to the protein (via primary amines, carboxyl groups, and the aromatic rings of tyrosine and histidine). These covalent binding methods were evaluated by immobilization of the enzyme alkaline phosphatase and with monoclonal antibodies tagged with 125I. It was determined that approximately 75% of the protein on the “covalently” immobilized samples was simply adsorbed. Adsorption was reduced by varying the pH and ionic strength of the wash buffers as well as by adding chaotropes and competitors to the wash buffer. A more efficient method of reducing adsorption was to perform the immobilization in the presence of a detergent; 0·5% Tween 60 was the most effective detergent studied. The optimized procedure gives a surface loading of 1·20 × 10 12 MAb/cm 2, of which approximately 10% was adsorbed protein. This surface density is consistent with a monolayer of immobilized protein. This protein was shown to be active by challenging immobilized antibodies with 35S-labelled antigen; only the antibody/antigen pair released radioactive material upon elution.

水‐有機溶媒二相反応系における牛脂の固定化リバーゼによる加水分解

Lipase from Candida cylindracea was immobilized on chitosan beads by covalent binding methods, and its application to hydrolysis of beef tallow was investigated in a biphasic organic-aqueous system. The addition of isooctane, found to be the best among the organic solvents tested, to the reaction system stimulated the hydrolysis of beef tallow by immobilized lipase strongly. The proportion of isooctane and aqueous buffer in the reaction mixture was chosen at the ratio of 1: 4. Under this condition, the reaction rate was greatly enhanced compared to that in the organic-solvent-free system, and the extent of hydrolysis reached to about 90%.

Hydrolysis of triglycerides by immobilized thermostable lipase from Humicola lanuginosa.

Thermostable lipase from Humicola lanuginosa No. 3 was immobilized by entrapment in photo-crosslinkable resin prepolymers, urethane prepolymers or Ca-alginate. Adsorption to different types of resins or gels, and covalent binding methods were also employed. From all the methods examined, adsorption on Amberlite XAD-7 followed by crosslinking with glutaraldehyde was superior, as to several points. Firstly, the adsorption efficiency for lipase was relatively high, being about 70%. Secondly, the optimal temperature was about 10°C higher than that for free lipase. The resin immobilized lipase was not affected by pH changes, and finally it also exhibited excellent stability as to repeated batch hydrolysis of triglycerides, both in a shaking flask system and in a recycling column reactor for more than 2 and 3 weeks, respectively.

Immobilization of polynucleotide phosphorylase on inorganic support

Polynucleotide phosphorylase was immobilized on aminopropyl porous glass by various covalent binding methods. The enzyme immobilized with glutaraldehyde exhibited the highest activity and had the same optimal pH as the free enzyme. The specific activity of the immobilized enzyme was increased by treatment with SH-reducing agent.