Immobilization Of Urease Research Articles

Polypyrrole/MWCNT nanobiocomposite based electrochemical urease biosensor

Fabrication of nanocomposite film of electrically conducting polypyrrole (PPy) and functionalized multi-walled carbon nanotubes (MWCNTs) on a stainless steel electrode by electro-deposition method and immobilization of urease onto the nanocomposite film to obtain a nanobiocomposite electrode as a sensitive electrochemical urease biosensor is reported. Cross-linking by glutaraldehyde (0.1%) method for the immobilization of urease (2 mg/mL) in a phosphate buffer solution of 0.1 molarity at a pH of 7.0 was used. The Characterization of the nanocomposite and nanobiocomposite film thus obtained was done by Scanning Electron Microscopy (SEM), Fourier Transform Infrared spectroscopy (FTIR), Cyclic Voltammetry (CV), and Electrochemical Impedance Spectroscopy (EIS). The increased size of the Cyclic voltammogram and shifting of anionic peaks towards the lower voltage indicates the incorporation of MWCNTs into the growing film during the electro-deposition of PPy on electrode. Reduction of the oxidation potential due to MWCNTs leads to lowering of potential for the electro-catalytic reduction of urea. The incorporation of functionalized MWCNT also made possible increased amount of enzyme concentration, an extended lifetime, long time stability and improved response times of the enzyme electrode. This modified nanobiocomposite electrode showed a good linear response to the urea concentration change in the range of 10 mM to 50 mM. The results obtained from Michaelis–Menten constant K´m, maximum current (Imax), detection limit, sensitivity, response time and shelf-life of electrochemical biosensor indicating good sensing for urea detection.

Laser-induced hierarchical carbon patterns on polyimide substrates for flexible urea sensors

Thermochemical decomposition of organic materials under heat-treatment in the absence of oxygen, known as the pyrolysis process, is often employed to convert micro and nano patterned polymers into carbon structures, which are subsequently used as device components. Pyrolysis is performed at ≥900 °C, which entails substrate materials with a high thermal stability that excludes flexible, polymeric substrates. We use optimized laser radiation to pattern graphitic carbon structures onto commercially available polyimide (Kapton) sheets in the micrometer to millimeter scale by inducing a localized, rapid pyrolysis, for the fabrication of flexible devices. Resulting laser carbon films are electrically conductive and exhibit a high-surface area with a hierarchical porosity distribution along their cross-section. The material is obtained using various combinations of laser parameters and pyrolysis environment (oxygen-containing and inert). Extensive characterization of laser carbon is performed to understand the correlation between the material properties and laser parameters, primarily fluence and power. A photothermal carbonization mechanism based on the plume formation is proposed. Further, laser carbon is used for the fabrication of enzymatic, pH-based urea sensors using two approaches: (i) direct urease enzyme immobilization onto carbon and (ii) electrodeposition of an intermediate chitosan layer prior to urease immobilization. This flexible sensor is tested for quantitative urea detection down to 10−4 M concentrations, while a qualitative, color-indicative test is performed on a folded sensor placed inside a tube to demonstrate its compatibility with catheters. Laser carbon is suitable for a variety of other flexible electronics and sensors, can be conveniently integrated with an external circuitry, heating elements, and with other microfabrication techniques such as fluidic platforms.

Immobilization of Urease on DEAE-Cellulose Strips for One Step Urea Detection

AbstractUrease isolated from pigeonpeas was immobilized on 1 cm × 6 cm diethylaminoethyl (DEAE)-cellulose strips. The optimum immobilization (55% activity) was observed at 4°C, with a protein concentration of 1 mg per strip at pH 6.5. Immobilized strips stored at 4°C showed an increased shelf-life and there was no leaching of enzyme from the immobilized strips for 6 months. Enzyme could be eluted on application of 0.2 M KCl in buffer, which showed that the binding of the enzyme was tight on DEAE-cellulose strips. These strips when used along with dye, phenol red (coimmobilized) gave different shades from light yellow to dark magenta depending upon the concentration of urea. The ease of immobilization of urease on DEAE-cellulose strips and easy availability of pigeonpeas urease described in the present study makes it a suitable product for potential applications in urea diagnostics.

Enzymatic Urea Sensor Based on Graphene Oxide/Titanium Dioxide Films Modified by Urease-Magnetic Beads

In this paper, we proposed an enzymatic urea sensor using urease-magnetic beads (MBs) modified graphene oxide (GO)/titanium dioxide (TiO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> ) composite layers. The urea sensor was prepared by screen-printing technology and radio frequency sputtering. TiO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> sensing film was deposited onto polyethylene terephthalate substrate, which serves as an efficient matrix for immobilization of urease. Preparation of GO/TiO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> films was completed after GO being deposited onto the TiO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> film. Moreover, the characterization and sensing properties of the urea sensor were performed by energy dispersive spectrometer, scanning electron microscope, atomic force microscopy, electrochemical impedance spectroscopy, and voltage-time measurement system. The results indicated that the GO/TiO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> urea sensor enhances its performance of average sensitivity, response time, and drift rate after modifying the sensing membrane though MBs.

Exuberant Immobilization of Urease on an Inorganic SiO2 Support Enhances the Enzymatic Activities by 3-fold for Perennial Utilization.

Urease has been covalently immobilized on a 3-D networking silica gel (SG) using dimethyldichlorosilane (DMDCS) as second generation silane coupling reagent and m-nitroaniline as linker component in a robust methodology and subsequently characterized as [{Si(OSi)4(H2O)0.05}205.2] n=4{OSi(CH3)2-NH-C6H4-N═N-urease}·282.5H2O (molecular mass 263 445 g or 263.4 kDa). Selective coupling of tyrosine residue with an identifiable m-nitroaniline modified SG unit prevents enzyme-enzyme cross-linking leading to enhancement of enzymatic activity. The material worked at room temperature and its activity (luminescent and ammonia releasing efficiency) was enhanced by 3-fold (for both synthetic and real sample) compared to native enzyme values at neutral pH. Up to 30 days and 30 cycles, this 3-fold activity remains as such but reduces gradually to native enzyme level after 60 days and 60 cycles of reuse.

Immobilization of urease in Langmuir-Blodgett films of di-ureasil hybrid compounds

Incorporation of enzymes in matrices as nanostructured films is a strategy to achieve molecular architectures able to enhance the efficiency of sensors and optoelectronic devices. In this present work, urease was inserted in the aqueous subphase of a di-ureasil hybrid compound (DUPTS) assembled as a Langmuir monolayer. Phosphotungstic acid (PWA) was introduced in the aqueous subphase in order to stabilize DUPTS by means of electrostatic interactions. Incorporation of urease in the Langmuir film could be attested with surface pressure and surface potential-area isotherms, rheological measurements, polarization modulation infrared reflection-absorption spectroscopy, and Brewster angle microscopy. The monolayers were transferred to solid supports as Langmuir-Blodgett films and characterized with infrared and fluorescence spectroscopies, proving not only the co-transfer of urease to the solid support, but also the conservation of part of its secondary structure. The enzyme activity could be detected towards urea hydrolysis enabling the urease/PWA/DUPTS supramolecular system to be employed for sensors as well as for other optoelectronic devices.

LED-Based Portable Optical Biosensor for Measurement of Serum Urea Levels Using Urease Immobilized Agarose-Guar Gum Composite Film

A simple, reliable, low cost, and portable biosensor has been developed for direct measurement of urea levels in serum samples. It is based on hydrolysis of urea by immobilized urease, generating ammonia that is estimated by modified Berthelot method using simple optical detection system. The immobilization of urease was achieved in agarose guar-gum composite film for the first time which offered an excellent retention of enzyme activity (90%–95%) with negligible leaching and good operational stability. The optical detection system involving LED light, optical fiber, and phototransistor provides simple and portable measurement system. The sensor shows linear response in the wide range of urea concentration from 0.025 to 10 mM with reusability for 10 cycles and superior storage stability of 180 days. The fabricated biosensor was successfully employed for measurement of normal and abnormal urea levels in serum samples with accuracy of 96% using very small sample volume ( $15~\mu \text{l}$ ). It showed an excellent correlation ( $r^{2}=0.99$ ) for determination of urea levels with that of using Siemens autoanalyzer.

RETRACTED ARTICLE: Effect of Urea on the Morphology of Fe3O4 Magnetic Nanoparticles and Their Application in Potentiometric Urea Biosensors

The effect of different concentrations of urea on the morphology of iron oxide (Fe3O4) magnetic nanoparticles was studied. Fe3O4 magnetic nanoparticles were fabricated by the coprecipitation method. The morphology, crystallinity, compositional purity, and emission characteristics were tested by the techniques of scanning electron microscopy (SEM), X-ray powder diffraction (XRD), and Raman characterization. The drop-casting technique was successfully used to fabricate a potentiometric urea biosensor producing initially isopropanol and chitosan solution, consisting of Fe3O4 nanoparticles, on a glass fiber filter. To measure the developed biosensor’s voltage signal from the functionalized nanoparticles, a copper wire was utilized. The Fe3O4 nanoparticle surface functionalization was performed through the electrostatic immobilization of urease with the Fe3O4-chitosan (CH) nanobiocomposite. The presented urea biosensor measured a wide logarithmic range of urea concentration of 0.1–80 mM with a sensitivity of 42 mV/decade, and indicated a fast response time of approximately 12 s. The developed urea biosensor showed enhanced sensitivity, stability, reusability, and specificity. All experimental results demonstrate the application potential of the developed urea sensor for the monitoring of urea concentrations in human serum, drugs, and food industry-related samples.

Spin‐coated ZnO–graphene nanostructure thin film as a promising matrix for urease immobilization of impedimetric urea biosensor

The fabrication, characterization, and application of a nanostructured zinc oxide/graphene (ZnO/Gr) transducer were described for the determination of urea concentration. For this purpose, thin films of sol–gel‐synthesized ZnO nanostructure and Gr were prepared using the spin coating technique on the conductive fluorinated–tin oxide (FTO) substrate. Appropriate correlations between the spinning parameters and thin film features were established so that an efficient ZnO–Gr thin film was obtained. Field emission scanning electron microscopy (FE‐SEM) images prove thin film pores to be an operative biosensing zone for urease (Urs) enzyme immobilization. Step‐by‐step monitoring of FTO/ZnO‐Gr/Urs biosensor fabrication was achieved using electrochemical methods. The impedimetric results of a fabricated FTO/ZnO‐Gr/Urs biosensor show high sensitivity for urea detection within 5.0–125.0 mg/dL and a limit of detection of 3.36 mg/dL.

Amino functionalization of carboxymethyl cellulose for efficient immobilization of urease

Jack bean urease (EC.3.5.1.5) was effectively immobilized onto amino functionalized epichlorohydrin cross-linked carboxymethyl cellulose (ECH-CMC) beads that were fabricated by graft co-polymerization of polyacrylamide (PAm) onto ECH-CMC beads in presence of potassium persulfate (KPS)/thiourea (TU) combined redox initiator followed by Hoffmann degradation. The progress of the synthesis along with immobilization processes was investigated by FTIR spectra. Also, the morphological structures of the beads before and after urease immobilizations were examined using SEM. Immobilization efficiency and immobilized urease amounts were estimated as a function of the amino functionalization degrees. The effects of pH and temperature on urease activity were studied. The results showed that after immobilization the optimum pH and temperature displayed higher shifts to 8 and 45°C, respectively, which reveal a higher structural stability upon immobilization performance. Moreover, the kinetic studies indicated that the urea hydrolysis reaction, which catalyzed by urease enzyme displayed a good fit with Michaelis–Menten equation, and the kinetic parameters Km and vm were estimated to be 14±0.7mM and 2±0.2μmolNH3/min·mg immobilized urease, respectively. Furthermore, the immobilized urease maintained approximately 88% of its initial activity after the 10th reuse cycle.

Immobilization of urease onto cellulose spheres for the selective removal of urea

Cellulose spheres were selected as an economical and biodegradable carrier for urease immobilization. The high crystallinity cellulose spheres (Ces) were crosslinked using citric acid and then oxidized using sodium periodate to produce aldehyde groups on the Ces surface that would bind covalently with the amino groups of urease through a Schiff base reaction. Ces, the obtained dialdehyde cellulose spheres (DACes) and cellulose spheres immobilized by urease (UrCes) were characterized by FTIR, XRD, SEM and TG. The results demonstrated the successful modification of Ces and immobilization of urease, along with the retention of the original spherical morphology and improved thermal stability. As an adsorbent, UrCes had both a greater adsorption capacity for urea and a greater specific selective adsorption than DACes. Urease was respectively immobilized on DACes with 19, 31 and 45% aldehyde contents to obtain UrCes1, UrCes2 and UrCes3. The maximum removal capacities of UrCes1, UrCes2, and UrCes3 for urea were 243.3, 276.2, and 187.6 mg g−1, while only 10.71, 15.08 and 20.50 mg g−1 l-phenylalanine were adsorbed, respectively.

Grafted Pectin with Glycidyl Methacrylate for Multi-Sites Urease Immobilization

Pectin poly glycidyl methacrylate copolymer (PGMA-g-pectin) was prepared via emulsion polymerization technique, characterized and used as multi-sites enzyme immobilization system. Urease, an enzyme model in this study, was sequentially immobilized onto the prepared carrier via both the carboxyl and epoxy groups. The structure and surface morphology of the prepared copolymer, before and after immobilization, were characterized by FT-IR and SEM. Both the amount of bounded urease and its relative activity were measured. A higher activity (about 68.4%) was measured for urease bounded to PGMA-g-SA activated with 10 mg of 1-[3-(dimethylamino) propyl]-3-ethylcarbo diimide hydrochloride (EDC) at pH 5 for 3 h. The various parameters affecting the potency of urease immobilization process (e.g. activation and immobilization time, pH and the concentration of EDC and urease) were investigated. The basic characteristics (optimum pH and temperature, thermal stability, storage stability and reusability) of the immobilized enzyme were also determined. The results showed that the immobilized urease maintained its excellent performance in detecting urea in 20 measurements and retained 70 % from its original activity after 60 days of storage at 4°C.

Extraction, purification, kinetic characterization and immobilization of urease from Bacillus sphaericus MTCC 5100

Abstract Urease is a well-known enzyme, that catalyzes the urea hydrolysis to ammonia and carbon dioxide. Industrial application of this enzyme in the beverages, fertilizer and pharmaceutical. In this research, microbial urease was purified, characterized and immobilized on metal electrode for biosensor application. Enzyme was purified step by step process, salt precipitation followed by ion exchange chromatography followed by gel filtration chromatography. The purification fold was achieved 6.67 with specific activity 32.5 IU. The estimated molecular weight and Km of the enzyme were 185 kDa and 2.0±0.5 mM respectively. SDS-PAGE was also performed to check the subunit of the purified enzyme and it was found to be three subunits 70 kDa, 60 kDa and 55 kDa. Immobilization of purified urease was performed by cross linking using glutaraldehyde on metal electrode for biosensor development. The storage stability was achieved up to 60 days when store at 4 °C in PBS buffer.

Investigating the Influence of Temperature on the Kaolinite-Base Synthesis of Zeolite and Urease Immobilization for the Potential Fabrication of Electrochemical Urea Biosensors.

Temperature-dependent zeolite synthesis has revealed a unique surface morphology, surface area and pore size which influence the immobilization of urease on gold electrode supports for biosensor fabrication. XRD characterization has identified zeolite X (Na) at all crystallization temperatures tested. However, N2 adsorption and desorption results showed a pore size and pore volume of zeolite X (Na) 60 °C, zeolite X (Na) 70 °C and zeolite X (Na) 90 °C to range from 1.92 nm to 2.45 nm and 0.012 cm3/g to 0.061 cm3/g, respectively, with no significant differences. The specific surface area of zeolite X (Na) at 60, 70 and 90 °C was 64 m2/g, 67 m2/g and 113 m2/g, respectively. The pore size, specific surface area and pore volumes of zeolite X (Na) 80 °C and zeolite X (Na) 100 °C were dramatically increased to 4.21 nm, 295 m2/g, 0.762 cm3/g and 4.92 nm, 389 m2/g, 0.837 cm3/g, in that order. The analytical performance of adsorbed urease on zeolite X (Na) surface was also investigated using cyclic voltammetry measurements, and the results showed distinct cathodic and anodic peaks by zeolite X (Na) 80 °C and zeolite X (Na) 100 °C. These zeolites’ molar conductance was measured as a function of urea concentration and gave an average polynomial regression fit of 0.948. The findings in this study suggest that certain physicochemical properties, such as crystallization temperature and pH, are critical parameters for improving the morphological properties of zeolites synthesized from natural sources for various biomedical applications.

DEVELOPMENT OF NEW BIOSENSOR FOR ARGININE DETERMINATION IN PHARMACEUTICALS

Arginine is a semi-essential amino acid, which means that there are biochemical ways for its biosynthesis, but in certain periods of life (e.g. at intense growth and development) and at some diseases the amount of amino acid biosynthesized in the organism is insufficient, therefore it should be provided by food. At present, pharmaceutical manufacturers offer a number of preparations based on the amino acid arginine. Unfortunately, there is no proper control of arginine concentration in the produced preparations, which results in the risk of the presence of counterfeit drugs in the pharmaceutical market. Therefore, the development of a portable device for determination of arginine concentration in pharmaceuticals is greatly needed.The aim of this work was the development of a new biosensor for arginine determination in pharmaceutical preparations.Methods: The conductometric method of analysis was used. The gold interdigitated electrodes deposited on sital substrate served as conductometric transducers. Two-layer co-immobilisation of urease and arginase on the transducer surface was performed by covalent cross-linking of enzymes with BSA by glutaraldehyde.Results: The most suitable method of urease immobilization was chosen to develop the technique of immobilization of both enzymes. The main parameters of the developed technique of immobilization (concentrations of both enzymes and GA, incubation time) were optimized. An influence of the buffer parameters (solution ionic strength, capacity and pH) on the biosensor operation was studied. The signals reproducibility and operational stability of the developed biosensor were investigated. Using the developed biosensor the arginine concentration was measured in some medications.Conclusions: A new conductometric biosensor based on two enzymes (arginase and urease) was developed for arginine determination. The developed biosensor can be successfully used to analyze the arginine concentration in pharmaceuticals. The obtained results correlated well with the values stated by the producers.

Two-dimensional ytterbium oxide nanodisks based biosensor for selective detection of urea

Herein, we demonstrate synthesis and application of two-dimensional (2D) rectangular ytterbium oxide (Yb2O3) nanodisks via a facile hydrothermal method. The structural, morphological, compositional, crystallinity, and phase properties of as-synthesized nanodisks were carried out using several analytical techniques that showed well defined 2D rectangular nanodisks/sheet like morphologies. The average thickness and edge length of the nanosheet structures were 20 ± 5nm and 600 ± 50nm, respectively. To develop urea biosensor, glassy carbon electrodes (GCE) were modified with Yb2O3 nanodisks, followed by urease immobilization and Nafion membrane covering (GCE/Yb2O3/Urease/Nafion). The fabricated biosensor showed sensitivity of 124.84μAmM−1cm−2, wide linear range of 0.05–19mM, detection limit down to ~ 2μM, and fast response time of ~ 3s. The developed biosensor was also used for the urea detection in water samples through spike-recovery experiments, which illustrates satisfactory recoveries. In addition, the obtained desirable selectivity towards specific interfering species, long-term stability, reproducibility, and repeatability further confirm the potency of as-fabricated urea biosensor.

Synthesis of 1,4-dihydropyrimidines with immobilized urease: effect of method immobilization on magnetic supports

AbstractThe effect of the urease immobilization method was studied on magnetic supports for the Biginelli/Hantzsch reaction. For this purpose, Fe

HO-1 and Nrf-2 deficiency affects generation and differentiation of induced pluripotent stem cells

The sensing element of biosensor is prepared by the immobilization of urease onto nano-structured polyaniline (PANi)-Nafion® (NSPN)/Au/Al2O3 synthesized by the chronopotentiometric technique. The sensitivity of lead ion at an amperometric urease based biosensor is obtained to be 743.5 µA ppm−1 for the linear dynamic range of 0.1–1.0 ppm Pb2+. The irreversible inhibiting kinetic models of the mercury and lead ions at the amperometric biosensors are proposed. The values of Kd (dissociation equilibrium constant of adsorbed type of enzyme and heavy metal ion) and kirr (irreversible inhibition reaction rate constant) for mercury and lead ions are obtained to be 0.45 ppm, 1.66 × 10−3 s−1, 8.67 ppm and 3.16 × 10−3 s−1, respectively. Compared with lead ion detection, the higher sensitivity to mercury ion of the biosensor is mainly caused by a higher inhibition efficiency (kirr/Kd).

Immobilization and characterization of urease onto different spacer arms attached magnetic nanoparticles

Immobilization and characterization of urease onto different spacer arms attached magnetic nanoparticles

Conjugated polymers nanostructured as smart interfaces for controlling the catalytic properties of enzymes

The search for new molecular architectures to improve the efficiency of enzymes entrapped in ultrathin films is useful to enhance the effectiveness of biosensors. In this present work, conjugated polymers, based on thiophene and fluorine, were investigated to verify their suitability as matrices for the immobilization of urease. The copolymer poly[(9,9-dioctylfluorene)-co-thiophene], PDOF-co-Th was spread on the air-water interface forming stable Langmuir monolayers as determined by surface pressure-area isotherms, polarization-modulation reflection-absorption infrared spectroscopy (PM-IRRAS), and Brewster angle microscopy (BAM). Urease was incorporated in the floating monolayers being further transferred to solid supports as mixed Langmuir-Blodgett (LB) films. These films were then characterized with transfer ratio, fluorescence spectroscopy, PM-IRRAS and atomic force microscopy, confirming the co-transfer of the enzyme as well as its structuring in β-sheets. The catalytic activity was detected for urease, with a lower reaction rate than that encountered for the homogeneous environment. This was attributed to conformational constraints imposed to the biomacromolecule entrapped in the polymeric matrix.