Covalent Immobilization Of Urease Research Articles

Screening and Identification of Herbal Urease Inhibitors Using Surface Plasmon Resonance Biosensor

Background and objectives: Urease, that catalyzes the hydrolysis of urea, has received substantial attention for its impact on living organisms’ health and human life quality. Urease inhibitors play important role in management of different diseases including gastritis and other gastrointestinal disorders. In the present study, a new surface plasmon resonance-based biosensor was designed to discover new urease inhibitors. Methods: The biosensor surface was prepared by the covalent immobilization of urease on carboxymethyldextran hydrogel (CMD 500D) via its primary amine groups. Results: The biosensor combined with an orthogonal enzyme inhibition assay was utilized for screening of 40 traditional medicinal plant extracts against Jack-bean urease. Among them, Laurus nobilis leaf extract displayed a high affinity with the immobilized urease; therefore, its active compound (quercetin) was isolated and identified as a urease inhibitor. The equilibrium constant (KD) and Gibbs free energy (ΔGbinding) values for the interaction of quercetin with urease were obtained to be 55 nM and -41.62 kJ/mol, respectively. The results of molecular docking analysis also confirmed our findings. Conclusion: This SPR-based biosensor represents a new, fast, reliable, and an accurate technique for the identification of new urease inhibitors or novel 'lead' compounds from natural resources.

Electrically Conducting Poly(Pyrrole-co-Para-Phenylenediamine) as Potentiometric Transducers in Urea Biosensor Fabrication

This research has focused on the fabrication of a urea biosensor based on electrically conducting poly (pyrrole-co-para-phenylenediamine) (PPy-co-PPD). High amount of free amino groups (-NH2), originating from PPD, in the PPy-co-PPD structures made them suitable for chemical immobilization of urease. The PPy-co-PPD films were prepared by one-step electrodeposition of the mixture of pyrrole and PPD. It was found that the morphology and conductivity of the PPy-co-PPD films were influenced by amount of PPD in the copolymer. Increasing amount of PPD in the copolymer led to decreasing electrical conductivity. Greater particle size and less packing were observed for the copolymer with high PPD content. XPS revealed the existence of free amino groups (-NH2) on the surface of PPy-co-PPD films. The PPy-co-PPD films were further subjected for covalent immobilization of urease, selective catalytic enzyme for urea. Potentiometric responses of the PPy-co-PPD films showed the highest sensitivity of 47.3-54.2 mV/pUrea (r2 > 0.99) over the urea concentration ranging from 0.5-10.0 mM (pUrea 2.0-3.3). Detection limits and response linearity were in the normal range of urea level. Response time was approximately 10 seconds. Leaching test revealed that the PPy-co-PPD film showed 83% reduction of urease leaching out of the PPy-co-PPD film during measurement, compared to the PPy film.

Enzymatic Electrode Obtained by Immobilizing of Urease into a Nanocomposite Film Based on Conducting Polymers and Different Additives

The nanocomposite film was obtained by electrochemical co-polymerization of the corresponding monomers in the presence of functionalized carbon nanotubes and different additives. By immobilizing urease on a modified electrode of type nanocomposite film/platinum substrate a novel amperometric biosensor for the detection of urea was prepared. The polymeric and nanocomposite films were utilized as conducting films for amperometric urea sensing after covalent immobilization of urease onto film-coated electrode. The electrochemical performance of the modified electrode was studied by cyclic voltammetry, chronoamperometry, and scanning electron microscopy. The immobilized urease on the nanocomposite film surface exhibited an excellent electrocatalytic response toward reduction urea. Amperometric response was measured as a function of urea concentration at a fixed potential, in this case the open circuit potential. The obtained sensor showed rapid response and good reversibility. The response time was less than 10 s, while the reversible time was about 5–15 s. The enzymatic nanostructured electrode exhibited high sensitivity, a wide linear range, and low detection limit for electrochemical detection of urea.

An amperometric urea bisosensor based on covalent immobilization of urease on N2 incorporated diamond nanowire electrode

N2 incorporated diamond nanowire (N-DNW) film electrochemical biosensor has utilized for the quantitative determination of urea in aqueous solution and urine sample. N-DNW electrode is wet-chemically cleaned (oxidation) by boiling in a mixture of H2SO4 and HNO3 (3:1) at 200°C for 2h to remove graphite. Urease (Urs) and glutamate dehydrogenase (GLDH) are covalently attached to the oxidized N-DNW electrode by activating the COOH group of N-DNW using ethyl(dimethylaminopropyl)carbodiimide as the coupling agent and N-hydroxysuccinimide as activator. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy data reveal that carboxylic and hydroxyl functionalized nature of N-DNW electrodes Urs–GLDH immobilized N-DNW (Urs–GLDH/N-DNW) has been successfully utilized in urea biosensor which exhibits good performance in sensitivity (6.18μA/mgdL/cm2), stability (~1 month), reproducibility, lower detection limit (3.87mg/dL) and fast response time (>10s). Urs–GLDH/N-DNW also exhibits electrochemical response when tested for different concentration of human urine in buffer solution (from 1:9 to 4:6). In addition, Urs–GLDH/N-DNW bioelectrode retains 80% of its initial enzyme activity for <1 month, when stored at 4–6°C in a refrigerator.

Amperometric urea biosensor based on platinum electrode modified with a nanocomposite film

A novel amperometric biosensor for the detection of urea was prepared by immobilizing urease on a modified electrode of nanocomposite film/platinum substrate. The nanocomposite film was obtained by electrochemical co‐polymerization of the corresponding monomers in the presence of functionalized carbon nanotubes and different additives. These nanocomposites were used as conducting films for amperometric urea sensing after covalent immobilization of urease onto nanocomposite‐coated electrodes. The electrochemical performance of the modified electrode was studied by cyclic voltammetry, chronoamperometry and scanning electron microscopy. The immobilized urease on the nanocomposite film surface exhibited an excellent electrocatalytic response toward the reduction of urea. Amperometric response was measured as a function of urea concentration at a fixed potential, in this case, the open circuit potential. The obtained sensor showed rapid response and good reversibility. The response time was less than 10 s, whereas the reversible time was approximately 5 to 15 s. The enzymatic nanostructured electrode exhibited high sensitivity, a wide linear range and low detection limit for the electrochemical detection of urea. Copyright © 2012 John Wiley &amp; Sons, Ltd.

An amperometric urea biosensor based on covalent immobilization of urease on copolymer of glycidyl methacrylate and vinylferrocene

A unique urea biosensor construction based on the direct covalent attachment of urease onto a polymeric electron transfer mediator, poly(glycidyl methacrylate-co-vinylferrocene)-coated electrode is described. Amperometric response was measured as a function of urea concentration, at a fixed potential of +0.35 V vs. Ag/AgCl in phosphate-buffered saline (pH 7.0). Covalent immobilization of the urease directly to the functionalized ferrocene copolymer surface produced biosensors with a short response time (about 3 s) and provided low detection limits. The stability, reusability, pH, and temperature response of the biosensor, besides its kinetic parameter, were also studied.

Covalent immobilization of urease on polysiloxane matrices containing 3-aminopropyl and 3-mercaptopropyl groups

A technique of covalent immobilization of urease on polysiloxane templates, involving the sol-gel method, based on the use of crosslinking reagents (such as glutaraldehyde and Ellman's reagent) has been proposed. Urease, covalently grafted onto the surface of a poly(3-mercaptopropyl)siloxane template, was shown to retain its activity (67-84%) and stability (a decrease of 10% was observed over a period of 300 days). Urease adsorbed onto the poly(3-mercaptopropyl)siloxane template exhibited a higher activity than the native enzyme. The 3-mercaptopropyl groups of the polysiloxane template could be brought into the vicinity of the active metal center of the adsorbed urease and start acting as proton donors, thereby increasing the rate of the reaction catalyzed by the enzyme. Covalent immobilization of urease onto a 3-aminopropyl-containing polysiloxane template was shown to be less efficient, because it resulted in considerable losses of the activity of the enzyme. Conversely, urease adsorbed onto this template exhibited a high activity (60-86%).

Immobilization of urease on cation-exchange membranes prepared by radiation-initiated graft copolymerization of acrylic acid on polyethene thin films

A covalent immobilization of urease was conducted on carboxylic cation-exchange membranes (CEM) prepared by radiation-initiated graft copolymerization of acrylic acid (AA) on polyethene (PE) thin films. Six types of CEM with different grafting degree (from 26.5 to 95.2%) were used as carry. The carboxyl groups were activated by the carbodiimide method in order to carry out a covalently immobilization. The amount of bound protein and the enzyme activity were determined in each immobilized system. It was established that the urease, immobilized on CEM with 64.2% grafting degree, featured the highest relative activity – 80.32%. The amount of bound protein on this membrane type was 6.01 mg/cm2. The basic characteristics of the immobilized and the free enzymes were determined (pHopt, Topt and pHstab). It was found out that the immobilized urease had greater thermal and storage stability in comparison with the free enzyme. It was proven that CEM with a grafting degree of 64.2% would be a suitable carrier for urease immobilization.

An amperometric urea biosensor based on covalent immobilization of urease onto an electrochemically prepared copolymer poly ( N-3-aminopropyl pyrrole-co-pyrrole) film

An amperometric biosensor has been developed for the quantitative determination of urea in aqueous solution. The principle is based on the use of pH-sensitive redox active dissolved hematein molecule. The enzyme, urease (Urs), was covalently immobilized on a conducting copolymer poly ( N-3-aminopropyl pyrrole-co-pyrrole) film, electrochemically prepared onto an indium-tin-oxide (ITO)-coated glass plate. The covalent linkage of enzyme and porous morphology of the polymer film lead to high enzyme loading and an increased lifetime stability of the enzyme electrode. Amperometric response was measured as a function of concentration of urea, at fixed bias voltage of 0.0 V vs. Ag/AgCl in a phosphate buffer (pH 7.0). The electrode gives a linear response range of 0.16–5.02 m M for urea in aqueous medium. The response time is 40 s reaching to a 95% steady-state current value, and 80% of the enzyme activity is retained for about 2 months.

Covalent immobilization of urease on polypyrrole microspheres for application as a urea biosensor

AbstractUrease has been covalently immobilized on polypyrrole microspheres chemically linked to conducting polypyrrole-polyvinyl sulfonate (PPY-PVS) films. These films were electrochemically prepared during 5 - 7 min at a constant current of 2 mA using indium - tin oxide (ITO) glass plates as the working electrode, and a standard calomel electrode as the reference electrode. Urease covalently linked to polypyrrole microspheres (by reaction of protein amino groups with aldehyde groups on the surface of the microspheres) was entrapped/adsorbed onto electrochemically prepared conducting PPY-PVS films deposited on ITO. Potentiometric measurements undertaken on these conducting polymer electrodes using an ammonium ion analyzer reveal that they can be used for estimating the urea concentration in solutions from 5·10-3 mol/l to 6·10-2 mol/l.