Determination of purine nucleosides and their bases by high-performance liquid chromatography using co-immobilized enzyme reactors

A selective and sensitive assay of inosine, guanosine, hypoxanthine, guanine and xanthine by high-performance liquid chromatography with immobilized enzyme reactors was developed. The separation was achieved on a Capcell Pak C18 column (15 cm x 0.46 cm I.D.) with a mobile phase of 0.1 M phosphate buffer (pH 8.0) containing 7 mM sodium 1-hexanesulphonate and 0.1 mM p-hydroxyphenylacetic acid. The fluorimetric detection of hydrogen peroxide using immobilized peroxidase and p-hydroxyphenylacetic acid was applied to the assay of these compounds, which were oxidized to yield hydrogen peroxide in the presence of immobilized enzyme (purine nucleoside phosphorylase, guanase and xanthine oxidase). Enzyme reactions occurred sufficiently without post-column addition of reagents. Enzymes that catalysed the conversion of purine compounds were co-immobilized on aminopropyl controlled-pore glass packed in stainless-steel tubing. The detection limits were 30-200 pg per injection.     

Simultaneous assay of hypoxanthine, xanthine and allopurinol by high-performance liquid chromatography and activation of immobilized xanthine oxidase as an enzyme reactor

A selective and sensitive assay of substrates (hypoxanthine, xanthine and allopurinol) of xanthine oxidase by reversed-phase liquid chromatography coupled with the use of immobilized enzyme reactors is described. These compounds were oxidized by immobilized xanthine oxidase and produced hydrogen peroxide, which was determined fluorometrically using immobilized peroxidase and p-hydroxyphenylacetic acid. The detection limits of hypoxanthine, xanthine and allopurinol were approximately 50, 120 and 130 pg per injection, respectively. Immobilized xanthine oxidase inhibited by oxipurinol during the assay was reactivated by 2,6-dichlorophenolindophenol and could be used for a long period without a significant activity loss. These methods were applied to plasma and urine samples.     

Enzyme sensors for determination of fish freshness

A simple and rapid procedure for the determination of fish freshness was developed and applied to the determination of the K(1) parameter (freshness indicator): K(1) = ([HXR] + [HX])/([IMP] + [HXR] + [HX]) x 100, where [IMP], [HXR] and [HX] are inosine monophosphate, inosine and hypoxanthine concentrations, respectively. A platinum electrode is used to detect hydrogen peroxide produced by the enzymatic reaction catalysed by xanthine oxidase immobilised on the electrode surface. The determination of inosine and inosine monophosphate was performed by the addition of nucleoside phosphorylase, 5'-nucleotidase or alkaline phosphatase to the buffer solution. Parameters such as type of buffer, amount of enzymes and sample treatment were optimised. With this procedure a linear response was obtained in the concentration range 1 x 10(-6)-2 x 10(-5)mol 1(-1) for hypoxanthine, inosine and inosine monophosphate. The detection limit was 5 x 10(-7) mol 1(-1).     

A Multiple Enzyme Method for Maasurement of K,Na ATPase

Abstract

A multiple enzyme method for spectrophotometrical measurement of K, Na ATPase activity is described. The method includes three enzymatic steps: Hydrolyses of ATP by ATPase, conversion of inosine to hypoxanthine and ribose -1- phosphate by purine nucleoside phosphorylase and finally xanthine oxidase mediated oxidation of hypoxanthine to xanthine with consequent formation of formazan from a tetrazolium salt. The phospholytic cleavage of inosine in the medium is dependent of an ATPase- phosphate complex.     

Development and application of a biosensor for hypoxanthine in fish extract

A hypoxanthine biosensor was constructed using immobilized xanthine oxidase and a polarographic electrode. The enzyme was covalently immobilized on a commercially available preactivated nylon membrane. The polarographic electrode detected hydrogen peroxide and uric acid released during the enzymatic reaction. The electrode responded linearly to hypoxanthine concentration in the range 3.6–107 μM. When applied to the determination of hypoxanthine in several fish meats, the results obtained agreed well with those obtained by the conventional enzymatic method. More than 40 assays could be performed with the same membrane and each sample could be assayed in ca. 2–3 min. The biosensor provides a reliable, simple, rapid and economical method for the measurement of hypoxanthine, a useful indicator of fish freshness.     

Electrochemical determination of xanthine and hypoxanthine in rat striatum with an acetylene black-dihexadecyl hydrogen phosphate composite film modified electrode by HPLC coupled with in vivo microdialysis

The fabrication and application of a new electrochemical detector for use in HPLC is presented. The detector consists of an electrode modified with a composite film composed of acetylene black and dihexadecyl hydrogen phosphate. The electrochemistry of xanthine and hypoxanthine at this chemically modified electrode (CME) was investigated by cyclic voltammetry. It is found that the CME exhibits efficient electrocatalytic activity towards xanthine and hypoxanthine, with good sensitivity, stability, and lifetime. The linear ranges cover three orders of magnitude, and the detection limits are 6.0 × 10^?8 mol L^?1 for xanthine and 2.5 × 10^?7 mol L^?1 for hypoxanthine (at an S/N ratio of 3). The method was coupled to in-vivo microdialysis sampling and successfully applied to quantify xanthine and hypoxanthine in rat striatal microdialysates of freely moving rats.     

A sensitive xanthine oxidase electrode with silk net for estimating fish freshness

An enzyme biosensor was constructed using a plate platinum electrode and immobilized xanthine oxidase (XOD).Only a very small quantity of enzyme was chemically immobilized on a special silk net.Hydrogen peroxide released during the enzymatic reaction was detected by the electrode at+0.65 V (vs.Ag/AgCl).The electrode was very sensitive to hypoxanthine and its detection limit was 1×10-7 mol/L.When it was applied to the determination of fish freshness,the results agreed well with those obtained by traditional methods-determination of total volatile basic nitrogen (TVB-N) and microbial count.A range for estimating the freshness of river fish was suggested.     

Amperometric detection of hypoxanthine and xanthine by enzymatic amplification using a gold nanoparticles-carbon nanohorn hybrid as the carrier

A novel gold nanoparticles-single-walled carbon nanohorn (GNPs-SWCNH) hybrid was synthesized for the construction of an amperometric biosensing platform. The GNPs-SWCNH hybrid was stable in aqueous solution for at least two weeks, and was characterized with scanning electron microscopy, transmission electron microscopy, and electrochemical impedance spectroscopy. The average diameter of GNPs in situ synthesized on the SWCNH was 5-8 nm, and the good interaction between GNPs and SWCNH was confirmed by ultraviolet-visible absorption spectroscopy. The GNPs-SWCNH immobilized on a platinum electrode showed high electrochemical activity toward the oxidation of hydrogen peroxide and uric acid with low applied potentials. Combining with the enzymatic reaction of xanthine oxidase (XOx), a biosensor for hypoxanthine and xanthine was constructed. The XOx-GNPs-SWCNH-based biosensor exhibited good responses to hypoxanthine and xanthine with the linear ranges of 1.5 to 35.4 and 2.0 to 37.3 μM, and the detection limits of 0.61 and 0.72 μM, respectively. The recovery test showed acceptable results. The gold nanoparticles functionalized carbon nanohorns provided a promising way to construct an electrochemical platform for sensitive biosensing.     

Development of A Dual Channel Chemiluminescence FIA System for Rapid Measurement of Fish Freshness

Abstract

A dual channel chemiluminescence FIA system for the rapid measurement of the fish freshness index KI $$ where [IMP], [HxR] and [Hx] are, respectively, the inosine 5′-monophosphate, inosine and hypoxanthine contents of fish meat was developed. The system consisted of a pair of chemiluminescence FIAs (CL-FIA); one for the measurement of [IMP] + [HxR] + [Hx], and the other for [HxR] + [Hx]. IMP, HxR or Hx were measured using a reactor comprising immobilized alkaline phosphatase, purine nucleoside phosphorylase (PNP) and xanthine oxidase (XOD). The calibration curves for IMP, HxR or Hx were linear from 0.06 μM to 100 μM for a sample volume of 20 μl. The time for a single KI measurement was less than 1 min. This fish freshness FIA system is rapid, convenient and applicable for fish freshness measurements in real fish samples.     

Disposable amperometric biosensors based on xanthine oxidase immobilized in the Prussian blue modified screen-printed three-electrode system

The screen-printed three-electrode system was applied to fabricate a new type of disposable amperometric xanthine oxidase biosensor. Carbon-working, carbon-counter and Ag/AgCl reference electrodes were all manually printed on the polyethylene terephthalate substrate forming the screen-printed three-electrode system by the conventional screen-printing process. As a mediator, Prussian blue could not only catalyze the electrochemical reduction of hydrogen peroxide produced from the enzyme reaction, but also keep the favorable potential around 0 V. The optimum operational conditions, including pH, potential and temperature, were investigated. The sensitivities of xanthine and hypoxanthine detections were 13.83 mA/M and 25.56 mA/M, respectively. A linear relationship was obtained in the concentration range between 0.10 μM and 4.98 μM for xanthine and between 0.50 μM and 3.98 μM for hypoxanthine. The small Michaelis-menten constant value of the xanthine oxidase biosensor was calculated to be 3.90 μM. The results indicate that the fabricated xanthine oxidase biosensor is effective and sensitive for the detection of xanthine and hypoxanthine.     

Amperometric Sensor and Immobilized Enzyme Electrode for the Determination of Enzymatic Activities

Abstract

A sensitive method for the rapid determination of activities of soluble or immobilized enzymes, based on the electrochemical detection of hydrogen peroxide is described. Kinetic studies (Vmax and K_M determinations) can be performed for all H₂O₂ generating enzymes (i.e. most of the oxidases) using an amperometric probe with a platinum anode at a fixed potential.

When associated with an immobilized glucose oxidase membrane, this sensor constitutes a glucose electrode and the activity of any hydrolase which releases glucose can be measured. There is no need for other auxiliary enzymes and no preincubation step is required. The possibility to carry out continuous analysis constitutes the main advantage of the described method.     

A Highly Sensitive Enzymatic Assay Method for Hypoxanthine Estimation Based on the Use of an Oxygen Electrode

Abstract

Using a Clark-type oxygen electrode as sensor, a highly sensitive enzymatic assay method for hypoxanthine was developed, based on xanthine oxidase. The sensitivity of the assay is comparable to that of standard chemiluminescent techniques. The incorporation of sulfite, 0 to 150 mM, allowed hypoxanthine determination over the range of 5 nM to 100 μM. The response time was 4 min or less at 30°C.     

Flow-injection determination of adenosine and inosine in blood plasma with immobilized enzyme columns connected in series and fluorimetric detection

A rapid and sensitive flow-injection method is described for the enzymatic determination of adenosine and inosine in human blood plama. Teflon columns prepared by packing adenosine deaminase. purine nucleoside phosphorylase, xanthine oxidase, uricase and horseradish peroxidase immobilized chemically on controlled-pore glass beads are connected in series in that order in the flow line. Hydrogen peroxide formed in the enzymatic conversion of adenosine and inosine is measured fluorimetrically after reaction with 3-(p-hydroxyphenyl)propionic acid. Linear calibrations were obtained for 0.5–500 pmol of adenosine or inosine in hte 20 μl sample injected. Necessary deproteination routines are outlined.     

Immobilization of xanthine oxidase and its use in the quantitation of hypoxanthine in fish muscle tissue extracts using a flow injection method

Fish muscle extracts (Scomberomorus— brasiliensis- carite) were analyzed for their hypoxanthine content using a flow injection system incorporating an immobilized xanthine oxidase bioreactor. The xanthine oxidase was immobilized under mild conditions to a 2-fluoro-1-methylpyridinium Fractogel support. The uric acid produced from the oxidation of hypoxanthine by the immobilized xanthine oxidase at pH 7.0 and 35‡C was monitored at 290 ran. Hypoxanthine concentrations as low as 4.4 Μmol/L can be detected. Up to 30 samples per hour can be analyzed at a flow rate of 1 mL/min, using 150 ΜL sample volumes and a bioreactor dimension of 1.0 cm x 2.0 mm id. Recovery yields were between 92 and 99%. Both within day and between day precisions gave CVs < 5.00% (n = 30). Good correlation (r = 0.998) is obtained when 78 fish samples were analyzed for their hypoxanthine content both by this FI method and a reference HPLC method.     

Mediated amperometric biosensor for hypoxanthine based on a hydroxymethylferrocene-modified carbon paste electrode

An amperometric enzyme electrode for the determination of hypoxanthine in fish meat is described. The hypoxanthine sensor was prepared from xanthine oxidase immobilized by covalent binding to cellulose triacetate and a carbon paste electrode containing hydroxymethylferrocene. The xanthine oxidase membrane was retained behind a dialysis membrane at a carbon paste electrode. The sensor showed a current response to hypoxanthine due to the bioelectrocatalytic oxidation of hypoxanthine, in which hydroxymethyiferrocene served as an electron-transfer mediator. The limit of detection is 6 × 10^?7 M, the relative standard deviation is 2.8% (n=28) and the response is linear up to 7 × 10^?4 M. The sensor responded rapidly to a low hypoxanthine concentration (7 × 10^?4 M), the steady-state current response being achieved in less than 1 min, and was stable for more than 30 days at 5 ° C. Results for tuna samples showed good agreement with the value determined by the conventional method.     

Electrochemical Determination of Superoxide Based on Cytochrome c Immobilized on DDAB‐Modified Powder Microelectrode

Abstract

Cytochrome c was immobilized at didodecyldimethylammonium bromide (DDAB)‐modified powder microelectrode and presented quasi‐reversible electrochemistry. The apparent surface coverage of cytochrome c is greatly enhanced by using powder microelectrode technique, which is 1.21×10^?8 mol/cm², more than one to three orders of magnitude larger than that obtained with thiol and DNA‐modified Au electrode. The cytochrome c modified powder microelectrode was applied for the amperometric determination of superoxide generated by the reaction of hypoxanthine with xanthine oxidase (XOD) in the presence of dioxygen.

The detection sensitivity of the modified powder microelectrode is 0.74 µA/cm² µM, which is larger than that reported in previous publications. The detection limit of the modified powder microelectrode (PME) is 0.5 µM, and the linear detection range is 0.86～5.93 µM (values of the concentration are all in terms of hypoxanthine concentration in the solution).     

Amperometric Determination of Lactic Acid. Applications on Milk Samples

Abstract

A hydrogen peroxide electrochemical sensor, coupled with immobilized lactate oxidase and covered with a cellulose acetate dialysis membrane has been applied in flow analysis of lactate in milk samples. The hydrogen peroxide produced by the enzymatic reaction is measured with a platinum electrode polarized at +650 mV versus Ag/AgCl. Milk samples were analyzed and compared with a spectophotometric reference method. The developed procedure is very simple and the short response time allows its use in assaying milk samples on dairy farms.     

Effect of iron chelates on luminol chemiluminescence in the presence of xanthine oxidase

Xanthine oxidase, in catalysing the oxidation of hypoxanthine to uric acid, produces hydrogen peroxide. Chemiluminescence is produced by oxidation of luminol by reactive hydroxyl radicals formed from H₂O₂ by Fe-EDTA and similar complexes. The concentrations of the various components in the active reagent are optimized in order to obtain a constant chemiluminescent signal of high intensity. The effect of chelate structure on chemiluminescence generation is studied, and a structure-activity relationship is deduced. The detection limit for xanthine oxidase is 5 pg.     

Characterization of mediated and non-mediated oxidase enzyme based glassy carbon electrodes

The performance and analytical characteristics of a glassy carbon glutaraldehyde immobilized glucose oxidase electrode have been established with regard to the direct detection of hydrogen peroxide produced from the reaction of glucose with oxygen. Measurements were performed at + 1.1 V vs. SCE, and selectivity was obtained by casting the surface with a cellulose acetate membrane. Results compared favorably with the classical platinum-enzyme probe. The mechanism of ascorbic acid interference in hydrogen peroxide detection is reported. Mediated detection was also investigated for oxidase enzymes (glucose oxidase and xanthine oxidase) immobilized on the bare glassy carbon electrode. The probes were characterized using a specific enzyme mediator in solution (phenazine methosulfate or dichlorophenol-indophenol) plus hexacyanoferrate(III) as an electrochemical mediator. The electrode was poised at + 0.36 V vs. SCE for the detection of hexacyanoferrate(II). The advantages of this dual mediator configuration include high stability and sensitivity of the electrochemical signal and the ability to use less positive potentials for increased selectivity. Application to other enzymes, such as hydrogenases, using such a binary redox configuration is suggested.     

Amperometric Determination of Galactose,Lactose and Dihydroxyacetone Using Galactose Oxidase in a Flow Injection System with Immobilized Enzyme Reactors and On-Line Dialysis

Abstract

A flow injection manifold containing a dialyzer and reactors with immobilized galactose oxidase and peroxidase was used for the determination of galactose in urine, lactose in milk and dihydroxyacetone in a biotechnological reaction medium. The hydrogen peroxide which is formed by the galactose oxidase reaction was detected by amperometric reduction of a mediator. The latter had been produced from hydrogen peroxide in a peroxidase catalyzed reaction. The hydrogen peroxide detection step was studied with several mediators and hexacyanoferrate (II) was selected. An ion exchange HPLC procedure was used to purify the galactose oxidase, in particular from catalase, and the kinetics and the selectivity of a reactor containing the immobilized enzyme was investigated. Columns for removal of certain interferents such as ascorbic acid were used in the determination of galactose in urine. The response to galactose standards was linear from the detection limit of 2 μM to 60 mM. The throughput was 45 samples per hour and the relative standard deviation 0.4%.