Simple optical fibre biosensor based on immobilised enzyme for monitoring of trace heavy metal ions

A simple optical fibre biosensor based on immobilised enzyme for monitoring of trace heavy metal ions has been developed. The biosensor recognition system was designed based on the inhibition of urease activity, where the urease is immobilised on ultrabind membrane. The studies of inhibition by the heavy metal ions Hg(II), Ag(I), Cu(II), Ni(II), Zn(II), Co(II) and Pb(II) were performed using a fibre-optic biosensor configuration, where the pH change resulting from the bio-catalytic hydrolysis of urea was monitored at the wavelength 615 nm spectroscopically, using commercial pH indicator strip before and after the exposure to the heavy metal ions. The immobilised urease was regenerated by l-cysteine. The linear response range between 1×10^-9–1×10^-5 M and the limit of detection 1×10^-9 M (0.2 g/L) for Hg(II) ions was achieved by employing the flow method. The optimisation of experimental parameters, including flow method, is also discussed.     

A New Metal-Chelated Cryogel for Reversible Immobilization of Urease

Poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) [poly(HEMA-GMA)] cryogel was synthesized by cryopolymerization technique at frozen temperature. Iminodiacetic acid (IDA) was then attached covalently to the cryogel as a chelating agent. Then, poly(HEMA-GMA)-IDA cryogel was chelated with Ni(II) ions and this novel metal affinity support was used for adsorption of urease from its aqueous solution. Urease adsorption experiments were carried out in a continuous system by using a peristaltic pump. Maximum urease adsorption onto poly(HEMA-GMA)-IDA-Ni(II) cryogel was found to be 11.30 mg/g cryogel at pH 5.0 acetate buffer and in 25 °C medium temperature. Urease adsorption capacity decreased with increasing ionic strength and increasing chromatographic flow rate. Adsorption kinetics of urease onto poly(HEMA-GMA)-IDA-Ni(II) cryogel was also investigated and it was found that Langmuir adsorption model is applicable for this adsorption study. This novel immobilized metal affinity chromatography support was used 10 times without any decrease at their adsorption capacity. It was also observed that urease enzyme was repeatedly adsorbed and desorbed without significant lost in enzymatic activity.     

A dip-and-read test strip for the determination of mercury(II) ion in aqueous samples based on urease activity inhibition.

A sensitive dip-and-read test strip for the determination of mercury in aqueous samples based on the inhibition of urease reaction by the ion has been developed. The strip has a circular sensing zone that containing two layers: the top layer is a cellulose acetate membrane where urease is immobilized on it; the bottom layer is a pH indicator wafer that is impregnated with urea. The principle of the measurement is based on the disappearance of a yellow spot on the pH indicator wafer. The elapsing time until the disappearance of the spot which depends on the concentration of mercury(II) ion is measured with a stopwatch. Under the experimental conditions, as low as 0.2 ng/ml mercury can be observed with the detection range from 0.2 to 200 ng/ml in water. Organomercury compounds give essentially the same response as inorganic mercury. Heavy-metal ions such as Ag(I), Cu(II), Cd(II), Ni(II), Zn(II), and Pb(II) as well as other sample matrixes basically do not interfere with the mercury measurement.     

Urease–glutamic dehydrogenase biosensor for screening heavy metals in water and soil samples

A screen-printed three-electrode amperometric biosensor based on urease and the nicotinamide adenine dinucleotide hydrogen (NADH)–glutamic dehydrogenase system was developed and applied to the screening of heavy metals in environmental samples. The development of an amperometric sensor for the monitoring of urease activity was feasible by coupling the urea breakdown reaction catalysed by urease to the reductive ammination of ketoglutarate catalysed by glutamic dehydrogenase (GLDH). The ammonia provided by the urea conversion is required for the conversion of ketoglutarate to glutamate with the concomitant oxidation of the NADH cofactor. NADH oxidation is monitored amperometrically at 0.3 V (vs. Ag/AgCl) after urease immobilization onto the screen-printed three-electrode configuration. Immobilization of urease on the surface of screen-printed electrodes was performed by entrapment in alginate gel and adsorption on the electrode in a nafion film. Low sensitivity to inactivation by metals was recorded after urease entrapment in alginate gel with detection limits of 2.9 and 29.8 mg L^–1 for Hg(II) and Cu(II), respectively. The use of the negatively charged nafion film created a more concentrated environment of cations in proximity to the enzyme, thus enhancing the urease inhibition when compared to gel entrapment. The calculated detection limits were 63.6 and 55.3 g L^–1 for Hg(II) and Cu(II), respectively, and 4.3 mg L^–1 for Cd(II). A significant urease inactivation was recorded in the presence of trace amounts of metals (g L^–1) when the enzyme was used free in solution. Analysis of water and soil samples with the developed nafion-based sensor produced inhibition on urease activity according to their metal contents. The obtained results were in agreement with the standard methods employed for sample analysis. Nevertheless, the use of the amperometric assay (with free urease) proved more feasible for the screening of trace amounts of metals in polluted samples.     

Electrical Control of Urease Activity Immobilized to the Conducting Polymer on the Carbon Felt Electrode

The activity of urease varies by its redox reaction. Active urease has an SH group that is essential to exhibit its activity, however, oxidation agents such as quinone compounds can oxidize the SH group in urease and a S–S bond is produced, resulting in the loss of enzyme activity. The reduction potential of cystine was almost the same as that of the recovery of urease activity. In this work, it has been found that the SH group of urease can be oxidized by not only chemical reaction but also by the direct electrode oxidation of urease and the produced S–S bond can be reduced to SH group by chemical and electrode reactions, and the original enzyme activity is recovered. This research shows that the regulation of urease activity is easily possible by changing the electrode potential of the porous carbon felt immobilized urease. The variation of urease activity was monitored by ammonia or carbon dioxide electrode equipped with the urease immobilized carbon felt, and the ammonia or carbon oxide generated from urea can transfer through the carbon felt to reach the each gas permeable membrane. The combination of gas electrode with porous conducting material such as carbon can supply the novel device for the electrochemical investigation of enzyme activity.     

Studies on catalytic fluorescence formation with peroxidase-like metallotetrakis(N-methylpyridiniumyl) porphyrins

The relative ability of peroxidase-like metallotetrakis(N-methylpyridiniumyl)porphyrins [Me-TMPyP, Me = Mn(III), Fe(III), Co(III), Ni(II), Cu(II), and Zn(II)] to catalyse the hydrogen peroxide oxidation of homovanillic acid to a fluorescent dimer has been studied. The complexes of Mn, Fe and Co are effective catalysts in the reaction, but the complexes of Ni, Cu and Zn are not. The catalytic behaviour of Mn-TMPyP, Fe-TMPyP and Co-TMPyP has been compared with that of HRP in both enzymatic and kinetic analysis. The sequence of peroxidase-like catalytic activity is Mn-TMPyP> Co-TMPyP> Fe-TMPyP. The catalytic activity of Mn-TMPyP is 84% of that of HRP. These Me-TMPyP (Me = Mn, Fe, and Co) compounds are good substitutes for HRP in enzymatic analysis. Traces of hydrogen peroxide and glucose can be determined with the Me-TMPyP systems.     

Synthesis and biological evaluation of Cu(II), Zn(II), and Ni(II) 3-(4-nitrophenyl)acrylic acid complexes with diamines as potential urease inhibitors

Five new Cu(II), Zn(II), and Ni(II) 3-(4-nitrophenyl)acrylic acid complexes were synthesized and evaluated for inhibitory activity on jack bean urease. All five complexes were structurally determined by single crystal X-ray analysis. Compared with the positive reference acetohydroxamic acid (IC₅₀?=?13.25?μM), Cu(II) complexes 3 and 4 showed the strongest inhibitory activity against jack bean urease (IC₅₀?=?1.23 and 1.17?μM). Ni(II) and Zn(II) complexes also exhibited inhibitory activities (IC₅₀?=?10.09–13.10?μM).     

Enhancement of Enzymatic IDE Biosensor Response Using Gold Nanoparticles. Example of the Detection of Urea

A new conductometric biosensor based on interdigitated electrodes (IDEs) has been developed for the detection of enzymatic substrates using gold nanoparticles (GNPs), synthesized bellowing the citrate process, with an average diameter of 23 nm and functionalized with urease using layer‐by‐layer technique. A detection limit of 100 µM of urea is obtained when cross‐linked urease is directly immobilized on top of the IDEs (interdigitated distance: 20 µm) whereas a detection limit of 2 µM is obtained when urease functionalized gold nanoparticles are deposited on the top of the IDEs. The use of gold nanoparticles allows the increase of the sensitivity of detection (from 10 µS/mM to 107 µS/mM) due to the decrease of the thickness of probed zone.     

醋酸纤维素膜固定化脲酶的研究

酶固定膜反应器兼具有反应和分离两种功能,是酶工程领域中较活跃的研究课题.随着酶固定化技术和水平的提高,各种固定化酶生物反应器不断涌现,其中以采用固定化脲酶技术制作的人工肾最为成功.     

Micro-enzyme field effect transistor sensor using direct covalent bonding of urease

Enzyme field effect transistors (ENFETs), which have been previously proposed for the detection of urea, consist of a pH ion-sensitive FET with urease enzyme immobilized in a polymeric membrane. A new means of preparing the enzymatic sensing layer is proposed in which urease is directly covalently bonded onto the silica insulator. The sensitivity, lifetime and response time of the ENFETs obtained are fairly good.     

Enzymatic activity and catalytic hydrogen evolution in reduced and oxidized urease at mercury surfaces

It was originally shown [10] that urease retains its enzymatic activity when adsorbed at bare mercury and solid amalgam surfaces. However the opinion later prevailed that, when adsorbed at bare metal electrodes, proteins are irreversibly denatured. Here we confirm that urease is enzymatically active at a bare solid amalgam surface as found by Santhanam et al., and we show that this enzyme is equally active at a thiol-modified amalgam surface. We also show that it is the reduced form of urease, which is enzymatically active at Hg surfaces. Oxidation of the protein, resulting in formation of disulfide bonds, strongly decreases the enzyme activity. Using constant current chronopotentiometric stripping (CPS) we show that the exposure of surface-attached urease to negative potentials results in the protein unfolding. The extent of the unfolding depends upon the amount of time for which the protein is exposed to negative potentials, and at very short times this unfolding can be avoided. At thiol-modified Hg surfaces the protein is less vulnerable to the effects of the electric field. We conclude that the loss of enzymatic activity, resulting from a 10 min exposure of the protein to −0.58 V, is not due to reduction of the disulfide bonds as suggested by Santhanam et al. This loss is probably a result of protein reorientation, due to reduction of the Hg-S bonds (formed by accessible cysteines), followed by prolonged electric field effect on the surface-attached protein.     

电导法测脲酶活性

电导法测脲酶活性朱元保，颜流水，何双娥（湖南大学化学化工系，长沙，４１００８２）关键词脲酶活性，电导法，米氏常数，活化能脲酶广泛存在于豆类中，脲酶活性已成为豆乳质量控制指标之一，目前测脲酶活性的常用方法有比色法、氨电极法和酸碱滴定法。本文根据脲酶催化...     

Development of bis-thiobarbiturates as successful urease inhibitors and their molecular modeling studies

Bis-thiobarbiturate derivatives 1–15 have been synthesized, characterized by1 HNMR and EI-MS and screened for urease inhibition. All compounds showed various degree of urease inhibitory activity with IC_(50) values ranging 7.45 0.12 74.24 0.81 mmol/L while the standard thiourea behaved normally(IC_(50) = 21.10 0.12). Compounds 1(IC_(50) = 7.45 0.12 μmol/L), 9(IC_(50) = 18.17 1.03 μmol/L) and 13(IC_(50) = 8.61 0.45 μmol/L) showed excellent urease inhibitory activity in the series. Molecular modeling studies were performed to understand the binding site with the bimetallic nickel center of the enzyme.Structure-activity relationship has also been established for these compounds. This study identified bisthiobarbiturate as a novel class of urease inhibitors.     

Development of urease based amperometric biosensors for the inhibitive determination of Hg (II)

Enzymatic amperometric procedures for measurement of Hg (II), based on the inhibitive action of this metal on urease enzyme activity, were developed. Screen-printed carbon electrodes (SPCEs) and gold nanoparticles modified screen-printed carbon electrodes (AuNPs/SPCEs) were used as supports for the cross-linking inmobilization of the enzyme urease. The amperometric response of urea was affected by the presence of Hg (II) ions which caused a decreasing in the current intensity. The optimum working conditions were found using experimental design methodology. Under these conditions, repeatability and reproducibility for both types of biosensors were determined, reaching values below 6% in terms of residual standard deviation. The detection limit obtained for Hg (II) was 4.2 × 10^?6 M for urease/SPCE biosensor and 5.6 × 10^?8 M for urease/AuNPs/SPCE biosensor. Analysis of the possible effect of the presence of foreign ions in the solution was performed. The method was applied to determine levels of Hg (II) in spiked human plasma samples.     

Determination of myoglobin based on its enzymatic activity by stopped-flow spectrophotometry

A new method has been developed for the determination of myoglobin (Mb) based on its enzymatic activity for the oxidation of o-phenylenediamine (OPDA) with hydrogen peroxide. Stopped-flow spectrophotometry was used to study the kinetic behavior of the oxidation reaction. The catalytic activity of Mb was compared to other three kinds of catalyst. The time dependent absorbance of the reaction product, 2,3-diamimophenazine (DAPN), at a wavelength of 426 nm was recorded. The initial reaction rate obtained at 40 degrees C was found to be proportional to the concentration of Mb in the range of 1.0 x 10(-6) to 4.0 x 10(-9)mol L(-1). The detection limit of Mb was found to be 9.93 x 10(-10)mol L(-1). The relative standard deviations were within 5% for the determination of different concentrations of Mb. Excess of bovine serum albumin (BSA), Ca(II), Mg(II), Cu(II), glucose, caffeine, lactose and uric acid did not interfere.     

Trace Level Analysis of Mercury Using Urease in Combination with an Ammonia Gas Sensitive Semiconductor Structure

Abstract

A method for the determination of mercury(II) ions at trace levels is described. The method is based on the profound inhibitory effect of mercury on the enzyme urease. The activity of the enzyme was determined by the rate of ammonia produced from urea as followed by an ammonia gas sensitive iridium thin metalfilm-oxide-semiconductor (IrTMOS) structure. Two systems were investigated. For the initial urease activity studies, a simple microcell was used. Also, a test plate, containing dry reagent strips with all necessary chemicals was developed, making the analytical procedure very simple to perform. The test volume applied was 2 μl and the sensitivity to standards of mercury(II) ions is at least 0.005 μM (1.0 ng/ml). One sample could be analyzed in less than 8 minutes. Furthermore, the kinetics of sensor response versus enzyme activity is discussed.     

Bipolar pulse conductometric detection of enzyme reactions in flow-injection systems Urea in serum and urine

Conductometric monitoring of enzymatic reactions in a flow-injection system is described. Two conductance cells are used and the responses are manipulated automatically to provide a differential signal. The hydrolysis of urea, catalyzed by urease, is used as an example to illustrate the technique. Blood serum and urine control standards are used to assess the precision and accuracy. The conductometric method is shown to be equal to, or better than existing methods for urea with regard to detection limits (0.1 mM in urine, 0.01 mM in serum), working range, precision (3% RSD), accuracy, and sample preparation. The method has a sample throughput of 20 h^?1.     

Kinetic parameters of urease immobilized on modified acrylonitrile copolymer membranes in the presence and absence of Cu(II) ions

Poly(acrylonitrile-methylmethacrylate-sodium vinylsulfonate) membranes were subjected to seven different chemical modifications and the amount of the newly formed groups was measured for each membrane. Urease was then covalently immobilized onto the modified membranes and the amount of bound protein was determined. The kinetic parameters V(max) and K(m) of the immobilized urease were studied under static and dynamic conditions. Results showed that the rate of the enzyme reaction was higher for the membranes modified with NH(2)OH . H(2)SO(4), NH(2)NH(2) . H(2)SO(4), NaOH + EDA and NaOH + GA + EDA. It was confirmed that the reaction rate, measured under dynamic conditions, was higher than that one determined under static conditions. The influence of Cu(II) ions, as inhibitors, on the enzyme reaction kinetics (V(i) and K(i)) was also investigated. It turned out that the most sensitive membranes towards Cu(II) were those modified with NH(2)NH(2) . H(2)SO(4), NaOH + EDA and H(2)O(2). The results initiated further investigations on the influence of other heavy metal ions (Cd(II), Zn(II), Ni(II) and Pb(II)) over urease bound to a NH(2)OH . H(2)SO(4)-modified membrane. It was found that the inhibition effect of the heavy metal ions over immobilized urease decreases in the order: Cu(II) > Cd(II) > Zn(II) > Ni(II) > Pb(II). [Diagram: see text]     

Enzyme and inhibition assay of urease by continuous monitoring of the ammonium formation based on capillary electrophoresis

We present here an easy‐to‐operate and efficient method for enzyme and inhibition assays of urease, which is a widely distributed and important enzyme that catalyzes the hydrolysis of urea to ammonia and CO₂. The assay was achieved by integrating CE technique and rapid on‐line derivatization method, allowing us to continuously drive the sample to the capillary, thus to measure the amount of the product ammonia from the beginning to the end of the reaction. The method exhibits excellent repeatability with RSD as low as 2.5% for the initial reaction rate (n = 5), with the LOD of ammonia of 20 μM (S/N = 5). The enzyme activity as well as the inhibition of urease by Cu²⁺ were investigated using the present method. The results show that Cu²⁺ is a noncompetitive inhibitor on urease, in accordance with the result published in the literature. The enzyme activity and inhibition kinetic constants were obtained and were found to be consistent with the results of traditional off‐line enzyme assays. Our study indicates that the present approach is a reliable and convenient method for analysis of the urease activity and inhibition kinetics by continuous on‐line monitoring of the ammonium formation based on CE.     

Thermodynamic study on the interaction of cyanide ion and jack bean urease at different temperatures

A method based on Isothermal Titration Calorimety (ITC) is described for the thermodynamic assay of jack bean urease. Inhibitory activity of cyanide ion was examined against jack bean urease (JBU), at 27 and 37 ^oC in 30 mM Tris buffer of pH = 7. The binding parameters of the CN^? + JBU complexation have been calculated. It was found that in the low and high concentrations of the cyanide ions, the JBU structure was destabilized, resulting in a decrease in its biological activity.