Characterization of DNA immobilized on electrochemically prepared conducting polypyrrole-polyvinyl sulfonate films

The article describes the adsorption characteristics of DNA onto electrochemically generated polypyrrole-polyvinyl sulfonate (PPY-PVS) films obtained as a function of pH. Adsorption on PPY doped with an anion proceeds by anion exchange, and since DNA possesses a fixed negative charge owing to PO ₄ ⁻ , it favors a very strong binding displacing PVS with favorable energetic interactions. Characterization of adsorbed DNA onto the PPY-PVS films was carried out by ultraviolet-visible, Fourier transform infrared spectroscopy, and cyclic voltammetric studies.     

Synthesis of glutamate by reductive amination of 2-oxoglutarate with the combination of hydrogenase and glutamate dehydrogenase

Glutamate synthesis by reductive amination of 2-oxoglutarate was performed by the combination of NADH regeneration system and glutamate dehydrogenase (GluDH). The conversion of 2-oxoglutamate to glutamate was 98% after 3 h, and the turnover number of NAD⁺was 17.     

Amperometric l-glutamate biosensor based on bacterial cell-surface displayed glutamate dehydrogenase

A novel l-glutamate biosensor was fabricated using bacteria surface-displayed glutamate dehydrogenase (Gldh-bacteria). Here the cofactor NADP⁺-specific dependent Gldh was expressed on the surface of Escherichia coli using N-terminal region of ice nucleation protein (INP) as the anchoring motif. The cell fractionation assay and SDS-PAGE analysis indicated that the majority of INP-Gldh fusion proteins were located on the surface of cells. The biosensor was fabricated by successively casting polyethyleneimine (PEI)-dispersed multi-walled carbon nanotubes (MWNTs), Gldh-bacteria and Nafion onto the glassy carbon electrode (Nafion/Gldh-bacteria/PEI-MWNTs/GCE). The MWNTs could not only significantly lower the oxidation overpotential towards NAPDH, which was the product of NADP⁺ involving in the oxidation of glutamate by Gldh, but also enhanced the current response. Under the optimized experimental conditions, the current–time curve of the Nafion/Gldh-bacteria/PEI-MWNTs/GCE was performed at +0.52 V (vs. SCE) by amperometry varying glutamate concentration. The current response was linear with glutamate concentration in two ranges (10 μM–1 mM and 2–10 mM). The low limit of detection was estimated to be 2 μM glutamate (S/N = 3). Moreover, the proposed biosensor is stable, specific, reproducible and simple, which can be applied to real samples detection.     

Surface acoustic wave sensor system applied to the kinetic study of urease in plant seeds

A new type of the surface acoustic wave (SAW) sensor system was delivered. Ure-ase from several kinds of plant seeds was extracted with different extracting solvents. The urease activity, Michaelis constant and other kinetic parameters were estimated for the first time by means of the new device-SAW sensor system. Some factors such as pH, temperature, activators and inhibitors are also discussed. The method can be applied to the determination of urea content in human urine and the experimental results consist with those reported.     

Immobilization of lactate dehydrogenase on tetraethylorthosilicate-derived sol-gel films for application to lactate biosensor

Tetraethylorthosilicate (TEOS)-derived sol-gel films were utilized for the immobilization of lactate dehydrogenase (LDH) by physical adsorption and sol-gel/LDH/sol-gel sandwich configuration. An attempt was made to ascertain the optimum pH and temperature for the immobilized LDH. It was shown that TEOS-derived sol-gel films containing physically adsorbed LDH exhibited linearity from 0.5 to 4 mM, whereas those containing LDH in sandwich configuration showed linearity from 0.5 to 3 mM l-lactate. These sol-gel films, immobilized with LDH, were found to be stable for about 4 weeks at 4–10°C.     

Photomodulation of glutamate dehydrogenase properties by red light

To gain some insight into the mechanism by which red light-biosystem interaction occurs, an investigation was made of certain features of purified glutamate dehydrogenase from beef liver (E.C. 1.4.1.3.) irradiated with either an He---Ne laser (632.8 nm) or a red light-emitting diode (650±20 nm). In both cases the energy dose was 0.24 J cm⁻². Significant changes in the glutamate dehydrogenase extinction coefficient measured at 275 nm, the capability of the enzyme to bind the reduced form of nicotinamide adenine dinucleotide (NADH), certain kinetic parameters, the pH and temperature dependence and the sensitivity to guanosine 5 triphosphate (GTP) and adenosine diphosphate (ADP) were found, probably due to the interaction of lightwith a protein domain containing a metal ion or ions. He---Ne laser and diode irradiation were found to differ with regard to their interaction with glutamate dehydrogenase. Interestingly, different effects were also found when an He---Ne laser and a non-coherent Xe---Hg lamp were used to irradiate glutamate dehydrogenase under the same experimental conditions. This confirms that non-coherent light at various power levels affects the isolated glutamate dehydrogenase.     

Smectic structures in electrochemically prepared poly(3,4‐ethylenedioxythiophene) films

Sub‐micrometer layers of electrochemically prepared methyl‐ and decyl‐substituted poly(3,4‐ethylenedioxythiophene) (PEDOT) carrying perchlorate counterions have been examined with grazing incidence X‐ray diffraction with synchrotron radiation. The materials were found to be partially crystalline, and the data could be ascribed to a model of sheets of π‐π stacked polymer chains with a smectic ordering of these sheets. An unsubstituted PEDOT sample with the polymeric polystyrenesulfonic acid as a counterion was also investigated and turned out to be essentially amorphous. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 945–952, 2003     

Amperometric glutamate biosensor based on self-assembling glutamate dehydrogenase and dendrimer-encapsulated platinum nanoparticles onto carbon nanotubes

A novel amperometric biosensor based on self-assembling glutamate dehydrogenase (GLDH) and poly(amidoamine) dendrimer-encapsulated platinum nanoparticles (Pt-PAMAM) onto multiwall carbon nanotubes (CNTs) has been developed for the determination of glutamate. The formation of the self-assembled (GLDH/Pt-PAMAM)_n/CNTs construction was investigated by ζ-potential and high resolution transmission electron microscopy (HRTEM). The results indicated the uniform growth of the layer-by-layer nanostructures onto carboxyl-functionalized CNTs. The electrocatalytic property of the (GLDH/Pt-PAMAM)_n/CNTs modified electrode to glutamate in presence of NAD⁺ (β-nicotinamide adenine dinucleotide, 0.1 mM) was investigated at a low overpotential 0.2 V by electrochemical measurements. The results showed it had series of attractive characteristics, such as a large determination range (0.2-250 μM), a short response time (within 3 s), a high sensitivity (433 μA/mM⁻¹ cm²) and good stability (85% remains after 4 weeks).     

Preparation of a potentiometric immobilized urease electrode and urea determination in serum

Polyvinylalcohol was activated with 2-fluoro-1-methylpyridiniumtoluene-4-sulphonate and urease (EC.3.5.1.5) was covalently linked to the activated matrix. PVA-urease was then immobilized on the surface of a pH glass electrode with gelatine gel and it was cross-linked using glutaraldehyde. This potentiometric membrane electrode provides a linearity to urea in the 8.910⁻⁵ to 1.110⁻³ M concentration range, but by changing the buffer concentration can be studied in the range of 10⁻⁴ to 10⁻² M urea concentration. Reproducibility experiments (n:20) were carried out with the urease enzyme electrode and with photometric methods for pooled serum sample. Average values for the two methods were 5.96 and 5.86 mM, variation coefficients were 2.5 and 3.5% respectively.     

Nylon tube-immobilized creatinine iminohydrolase and glutamate dehydrogenase in serum and urine creatinine analysis

Immobilized enzyme nylon-tube reactors incorporating creatinine iminohyrolase (CI) and glutamate dehydrogenase (GDH) were used to assay creatinine in serum and urine. Optimum substrate concentrations for the assay were determined. The reactors were incorporated into a continuous flow system for creatinine analysis. The method was evaluated with respect to linearity, sample interaction, precision, accuracy, and analytical recovery. Comparison studies were carried out with a standard Jaffé method and the effect of interfering substances was investigated. From the results obtained, it was concluded that the assay was suitable as a simple, reliable, and specific method for serum and urine creatinine determinations.     

Characterization of urease derived from Citrullus lanatus (watermelon) seeds to estimate total Kjeldahl nitrogen in human urine

A urease extract prepared by decanting liquid from a suspension of finely ground Citrullus lanatus (watermelon) seeds was characterized and applied to dilute urine samples to demonstrate a low-cost field method to estimate total Kjeldahl nitrogen (TKN) concentrations in human urine. The extract exhibited a Michaelis-Menten constant, K_m, of 3.00 mM urea and a specific activity of up to 12.2 U/mg protein at an optimum pH of 8.1. A statistical F-test on 54 samples demonstrated that TKN can be estimated as the total ammonium-nitrogen recovered upon addition of urease in dilute fresh and stale urine samples. The total ammonium-nitrogen in urine samples determined after treatment with watermelon seed urease was consistent with that determined using traditional acid digestion techniques. The extract retained 85% of its initial capacity after three months of refrigeration. The effectiveness of this method to assay nitrogen in unbuffered urine samples will be useful in nitrogen analyses in nutrient recovery and urine or slurry storage contexts. Accordingly, this study is useful in understanding the kinetics of a plant-derived urease acting in dilute urine.     

The O to S substitution in urea brings inhibition activity against thiocyanate dehydrogenase

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壳聚糖固定化脲酶的制备和废水中的尿素分析

流动注射;尿素测定;壳聚糖固定化脲酶的制备和废水中的尿素分析     

The enzymatic determination of mercury and copper using acid urease. The effects of buffers

We report on a quick and simple test based on enzyme inhibition for the detection of mercury and copper using free acid urease coupled to an optical sensor system. Lipophilized Nile Blue was incorporated in plasticized poly(vinyl chloride) (PVC) to produce an ammonium-sensitive layer with a thickness of around 4 m. The layer was fixed on one side of a disposable cuvette. A solution of buffer, enzyme and heavy metals was placed into the thermostated cell. Enzymatic hydrolysis was started upon addition of urea and the formation of ammonium was monitored. Mercury and copper were the strongest inhibitors; for this reason the inhibitory efficiency of these metals was examined in citrate, acetate and trismaleate buffers. The cuvette test was most sensitive and selective for mercury in a citrate buffer. The limit of detection for mercury(II) ions was as low as 1 g/L. Copper ions do not interfere because of complexation by citrate. The inhibitory effects of metal combinations on the activity of acid urease and the effects of optimum pH of the enzyme and the transducer on the dynamic range of the cuvette test are presented.     

Activity and lifetime of urease immobilized using layer-by-layer nano self-assembly on silicon microchannels

Urease has been immobilized and layered onto the walls of manufactured silicon microchannels. Enzyme immobilization was performed using layer-by-layer nano self-assembly. Alternating layers of oppositely charged polyelectrolytes, with enzyme layers “encased” between them, were deposited onto the walls of the silicon microchannels. The polycations used were polyethylenimine (PEI), polydiallyldimethylammonium (PDDA), and polyallylamine (PAH). The polyanions used were polystyrenesulfonate (PSS) and polyvinylsulfate (PVS). The activity of the immobilized enzyme was tested by pumping a 1 g/L urea solution through the microchannels at various flow rates. Effluent concentration was measured using an ultraviolet/visible spectrometer by monitoring the absorbance of a pH sensitive dye. The architecture of PEI/PSS/PEI/urease/PEI with single and multiple layers of enzyme demonstrated superior performance over the PDDA and PAH architectures. The precursor layer of PEI/PSS demonstrably improved the performance of the reactor. Conversion rates of 70% were achieved at a residence time of 26 s, on d 1 of operation, and >50% at 51 s, on d 15 with a six-layer PEI/urease architecture.     

Fluorometric determination of urea in alcoholic beverages by using an acid urease column-FIA system

An acid urease column was applied to a fluorometric flow-injection analysis (FIA) system as a recognition element for determination of urea in rice wines.

The acid urease has specific properties of showing its catalytic activity in low pH range and tolerance to ethanol in comparison to those of a urease from jack-beans. The enzymes were covalently immobilized onto porous glass beads with controlled pore size and then, packed into a small polymer column. The flow-type of the biosensing system was assembled with a sample injection valve, the immobilized enzyme column, and a flow-through quartz cell attached to a fluorescent spectrophotometer. Citrate buffer (50 mM, pH 5.0) as the carrier solution was continuously pumped through the system. Sample solutions were introduced into the system via a rotary injection valve. A standard urea solution was measured through monitoring variations in fluorescent intensity attributable to fluorescent isoindole derivatives formed by coupling with ammonia molecules released in the enzymatic hydrolysis of urea and orthophthalaldehyde reagents. The fluorescent intensity was measured under the conditions of λ_ex = 415 nm and λ_em = 485 nm. A wide, linear relationship was obtained between the concentration of urea (1.0–100 μM) and the variation in fluorescent intensity. The monitoring did not suffer from ethanol and various amino acids contained in rice wines. Real samples pretreated with ion exchange resins for removal of endogenous ammonia were introduced into the FIA system and urea in the samples was determined. These results were compared with those obtained with use of an F-kit method. The proposed FIA system should present sensitive, selective and convenient analysis of urea in alcoholic beverages.     

Mathematical Model and Numerical Simulation of Conductometric Biosensor of Urea

In this article, a mathematical model was developed to describe and optimize the configuration of the urea biosensor. The biosensor is based on interdigitated gold microelectrodes modified with a urease enzyme membrane. The model presented here focuses on the enzymatic reaction and/or diffusion phenomena that occur in the enzyme membrane and in the diffusion layer. Numerical resolution of differential equations was performed using the finite difference technique. The mathematical model was validated using experimental biosensor data. The responses of the biosensor to various conditions were simulated to guide experiments, improve analytical performance, and reduce development costs.     

Solid state structural aspects of electrochemically prepared poly (p -phenylene) thin films – crystalline order and spherulite morphology

The electrochemical polymerization of benzene via the microemulsion approach yields highly crystalline and anisotropic “spherulitic” polyparaphenylene (PPP) thin films. The crystalline order and the origin of spherulite morphology are discussed. Received: 29 July 1997 / Accepted: 27 October 1997     

Immobilization of urease on composite fibre by using a gel formation of cellulose acetate and titanium iso-propoxide

Urease was entrap-immobilized on composite gel fibre of cellulose acetate and TiO₂. The immobilization can be easily performed under mild conditions. The gel fibre is stable in high ionic solutions and in phosphate solutions with a range of pH 6–8.The pH and thermal stabilities of immobilized urease were higher than those of the native one.