Miniaturized amperometric biosensor based on xanthine oxidase for monitoring hypoxanthine in cell culture media

Fabrication and characterization of miniaturized amperometric hypoxanthine biosensors are described and demonstrated for monitoring hypoxanthine in myocardial cell culture media. The sensors are based on xanthine oxidase (XO) immobilized on carbon fiber microelectrodes (CFMEs) using a composite film of Nafion and electropolymerized phenol (PPh). Nafion was used for XO immobilization because of its film hydrophobicity, enzyme-favored environment, and electrostatic interaction with XO, which was dispersed in Nafion film by immersing the Nafion-coated CFMEs in XO solution for 5 h. PPh film was formed as an overlay on Nafion and XO-modified CFMEs via electropolymerization. Hypoxanthine was measured with the sensor by the oxidation of enzymatic reaction products, hydrogen peroxide (H(2)O(2)), and uric acid (UA) at +0.60 V (vs Ag/AgCl). The use of Nafion and PPh as a matrix for XO immobilization yields enhanced specificity, sensitivity, and linearity toward hypoxanthine. A dynamic linear range of 5.0 microM to 1.8 mM was achieved with a calculated detection limit of 1.5 microM (S/N = 3) and a sensitivity of 3.144 nA/mM. In addition, the measurement was virtually interference-free from easily oxidizable species such as UA, ascorbic acid, physiological levels of neurotransmitters, and their principal metabolites. The biosensor was used to monitor hypoxanthine accumulation in myocardial cell culture media, in which the level of extracellular hypoxanthine was found to increase with ischemic tolerance.     

Optimization of a polypyrrole glucose oxidase biosensor

An amperometric glucose biosensor was fabricated by the electrochemical polymerization of pyrrole onto a platinum electrode in the presence of the enzyme glucose oxidase in a KCl solution at a potential of + 0·65 V versus SCE. The enzyme was entrapped into the polypyrrole film during the electropolymerization process. Glucose responses were measured by potentio-statting the enzyme electrode at a potential of + 0·7 V versus SCE in order to oxidize the hydrogen generated by the oxidation of glucose by the enzyme in the presence of oxygen. Experiments were performed to determined the optimal conditions of the polypyrrole glucose oxidase film preparation (pyrrole and glucose oxidase concentrations in the plating solution) and the response to glucose from such electrodes was evaluated as a function of film thickness, pH and temperature. It was found that a concentration of 0·3 M pyrrole in the presence of 65 U/ml of glucose oxidase in 0·01 M KCl were the optimal parameters for the fabrication of the biosensor. The optimal response was obtained for a film thickness of 0·17 μm (75 mC/cm²) at pH 6 and at a temperature of 313 K. The temperature dependence of the amperometric response indicated an activation energy of 41 kJ/mole. The linearity of the enzyme electrode response ranged from 1·0 mM to 7·5 mM glucose and kinetic parameters determined for the optimized biosensors were 33·4 mM for the K_m and 7·2 μA for the I_max. It was demonstrated that the internal diffusion of hydrogen peroxide through the polypyrrole layer to the platinum surface was the main limiting factor controlling the magnitude of the response of the biosensor to glucose. The response was directly related to the enzyme loading in the polypyrrole film. The shelf life and the operational stability of the optimized biosensor exceed 500 days and 175 assays, respectively. The substrate specificity of the entrapped glucose oxidase was not altered by the immobilization procedure.     

An FIA biosensor system for the determination of phosphate.

A flow injection analysis (FIA) biosensor system for the determination of phosphate was constructed using immobilized nucleoside phosphorylase and xanthine oxidase and an amperometric electrode (platinum vs silver/silver chloride, polarized at 0.7 V). When a phosphate-containing sample was injected into the detection cell, phosphate reacted with inosine in the carrier buffer to produce hypoxanthine and ribose-1-phosphate in the presence of nucleoside phosphorylase. Hypoxanthine was then oxidized by xanthine oxidase to uric acid and hydrogen peroxide, which were both detected by the amperometric electrode. The response of the FIA biosensor system was linear up to 100 microM phosphate, with a minimum detectable concentration of 1.25 microM phosphate. Each assay could be performed in 5-6 min and the system could be used for about 160 repeated analyses. This system was applicable for the determination of phosphate in various food products and plasma, and the results obtained agreed well with those of the enzymatic assay.     

Development of mediated amperometric biosensors for hypoxanthine, glucose and lactate: a new format

Electrochemical growth was used to form the organic conducting salt of tetrathiafulvalene (TTF) and tetracyanoquinodimethane (TCNQ) on platinum wires inserted in a glass capillary. Glucose oxidase, lactate oxidase and xanthine oxidase were deposited and crosslinked on the salt structure to produce mediated biosensors responsive to the corresponding analytes. Reliability, stability, interference, and the effect of oxygen on the electrode's response were studied. Among three common electroactive interfering substances tested, ascorbic acid was very active at the TTF-TCNQ structure and the highest response was exhibited by the enzyme-free electrode. Acetaminophen and uric acid displayed similar behaviour at a lower magnitude. The presence of oxygen significantly decreased the current responses of all electrodes.

The xanthine oxidase-bearing mediated electrodes were able to assay the hypoxanthine content of either the fish extract, fish homogenate or slurry of manually ground tissue, yielding results in good agreement with conventional enzymatic assays. The electrodes were stable more than 120 days and could be reused more than 30 times without losing their original activities.     

Improved selectivity and stability of glucose biosensor based on in situ electropolymerized polyaniline-polyacrylonitrile composite film

A new type of in situ electropolymerization method was used for electrochemical biosensor design. The biologic film was prepared by in situ electropolymerization of aniline into microporous polyacrylonitrile-coated platinum electrode in the presence of glucose oxidase. The novel glucose biosensor exhibited good selectivity, sensitivity and stability, which showed no apparent loss of activity after 100 consecutive measurements and intermittent usage for 100 days with storage in a phosphate buffer at 4 degrees C. Blood glucose determinations agreed well with standard hospital laboratory analysis. The construction and operational parameters of the biosensor were also optimized.     

Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O2.- and H2O2 during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-beta-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O2 consumption, only slightly but substantially inhibited in a competitive manner the O2.- -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Application of Chemiluminescence of a Cypridina Luciferin Analog to Immobilized Enzyme Sensors

Chemiluminescence of a Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydro-imidazo[l,2-a]pyrazin-3-one, was applied to immobilized enzyme sensors. Xanthine oxidase, peroxidase, glucose oxidase, uricase and cholesterol oxidase were immobilized by using photo-crosslinkable resin prepolymer or ion-exchangeable cellulose beads. The immobilized enzyme sensor system was composed of a photoncounter and a test tube in which the immobilized enzyme membrane or particles were placed. A linear relation between the concentration of substrates and luminescence rate was obtained on a logarithmic scale. This immobilized enzyme sensor system could be used repeatedly. Hydrogen peroxide, xanthine and hypoxanthine were measured sensitively and rapidly within 100 sec. Glucose, cholesterol and uric acid were measured sensitively within 10 min but could be measured within 100 sec, although less sensitive. The detection limits for xanthine, hypoxanthine, hydrogen peroxide, glucose, cholesterol and uric acid were 0.02, 0.02, 0.2, 0.4, 2 and 2 μM, respectively. Concentrations of hypoxanthine in tuna muscle, and glucose and cholesterol in serum measured using this sensor system were comparable with those measured by the standard methods.     

Bienzymatic amperometric biosensor for choline based on mediator thionine in situ electropolymerized within a carbon paste electrode

An amperometric enzyme biosensor for the determination of choline utilizing two enzymes, choline oxidase (CHOD) and horseradish peroxidase (HRP), is described. The biosensor consisted of CHOD cross-linked onto a HRP-immobilized carbon paste electrode. The biosensor was prepared by in situ electropolymerization of poly(thionine) within a carbon paste containing the enzyme HRP and thionine monomer and then CHOD was immobilized by using chitosan film through cross-linking with glutaraldehyde. The in situ electrogenerated poly(thionine) displays excellent electron transform efficiency between the enzyme HRP and the electrode surface, and the polymer enables improvement in enzyme immobilization within the paste. Several parameters such as the amount of thionine and enzyme, the applied potential, the pH, etc. have been studied. Amperometric detection of choline was realized at an applied potential of -0.2V vs saturated calomel electrode in 1/15M phosphate buffer solution (pH 7.4) with a linear response range between 5.0 x 10(-6) and 6.0 x 10(-4)M choline and a response time of 15s. When applied to the analysis of phosphatidylcholine in serum samples, a 0.997 correlation was obtained between the biosensor results and those obtained by a hospital method.     

Posttranslational ruling of xanthine oxidase activity in bovine milk by its substrates

The aims of this study were to test the hypothesis that the substrates of xanthine oxidase (XO), xanthine and hypoxanthine, are consumed while the milk is stored in the gland between milkings, and to explore how XO activity responds to bacteria commonly associated with subclinical infections in the mammary gland. Freshly secreted milk was obtained following complete evacuation of the gland and induction of milk ejection with oxytocin. In bacteria-free fresh milk xanthine and hypoxanthine were converted to uric acid within 30 min (T1/2 approximately 10 min), which in turn provides electrons for formation of hydrogen peroxide and endows the alveolar lumen with passive protection against invading bacteria. On the other hand, the longer residence time of milk in the cistern compartment was not associated with oxidative stress as a result of XO idleness caused by exhaustion of its physiological fuels. The specific response of XO to bacteria species and the resulting bacteria-dependent nitrosative stress further demonstrates that it is part of the gland immune system.     

Development of a biosensor for assaying postmortem nucleotide degradation in fish tissues

An enzyme sensor system has been developed to assess the freshness level in fish tissue. The system was designed to measure the K value, the concentration ratio of [Hx + HxR] and [Hx + HxR + IMP], where Hx, HxR, and IMP are hypoxanthine, inosine and inosine-5'-monophosphate, respectively. The [Hx + HxR] concentration in tissue extract was measured by nucleoside phosphorylase and xanthine oxidase immobilized on a preactivated nylon membrane and attached to the tip of a polarographic electrode. The electrode amperometrically detected the products of degradation, hydrogen peroxide and uric acid. For determination of [IMP + HxR + Hx], IMP was first converted to HxR by nucleotidase immobilized on the wall of a polystyrene tube. The enzyme electrode consisting of nucleoside phosphorylase and xanthine oxidase provided excellent reproducible results for at least 40 repeated assays and immobilized nucleotidase was good for at least 40 assays as well. The K value for each sample could be determined in ca. 10 min. When applied to K value measurements in several fish meats, the results obtained agreed well with those obtained by the conventional enzymatic method.     

Purification and characterization of a novel caffeine oxidase from Alcaligenes species

Alcaligenes species CF8 isolated from surface water of a lake produced a novel serine type metallo-caffeine oxidase. The optimal medium for caffeine oxidase production by this strain was (w/v) NaNO(3), 0.4%; KH(2)PO(4), 0.15%; Na(2)HPO(4), 0.05%; FeCl(3).6H(2)O, 0.0005%; CaCl(2).2H(2)O, 0.001%; MgSO(4).7H(2)O, 0.02%; glucose, 0.2%; caffeine, 0.05%, pH 7.5. The enzyme was purified to 63-fold by using ammonium sulfate precipitation, dialysis, ion exchange (diethylaminoethyl-cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified caffeine oxidase was monomeric with a molecular mass of 65 kDa. The purified caffeine oxidase with a half-life of 20 min at 50 degrees C had maximal activity at pH 7.5 and 35 degrees C. The purified caffeine oxidase had strict substrate specificity towards caffeine (K(m) 8.94 microM and V(max) 47.62 U mg protein(-1)) and was not able to oxidize xanthine and hypoxanthine. The enzyme activity was not inhibited by para-chloromercuribenzoic acid, iodoacetamide, n-methylmaleimide, salicylic acid and sodium arsenite indicating the enzyme did not belong to xanthine oxidase family. The enzyme was not affected by Ca(+2), Mg(+2) and Na(+), but was completely inhibited by Co(+2), Cu(+2) and Mn(+2) at 1mM level. The novel caffeine oxidase isolated here from Alcaligenes species CF8 may be useful in biotechnological processes including waste treatment and biosensor development.     

Construction and application of an amperometric xanthine biosensor based on zinc oxide nanoparticles-polypyrrole composite film

Zinc oxide nanoparticles (ZnO-NPs) were synthesized from zinc nitrate by simple and efficient method in aqueous media at 55°C without any requirement of calcinations step. A mixture of ZnO-NPs and pyrrole was eletropolymerized on Pt electrode to form a ZnO-NPs-polypyrrole (PPy) composite film. Xanthine oxidase (XOD) was immobilized onto this nanocomposite film through physiosorption. The ZnO-NPs/polypyrrole/Pt electrode was characterized by Fourier transform infrared (FTIR), cyclic voltammetry (CV), X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and electrochemical impedance spectroscopy (EIS) before and after immobilization of XOD. The XOD/ZnO-NPs-PPy/Pt electrode as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode were connected through a potentiostat to construct a xanthine biosensor. The biosensor exhibited optimum response within 5s at pH 7.0, 35°C and linearity from 0.8 μM to 40 μM for xanthine with a detection limit 0.8 μM (S/E=3). Michaelis Menten constant (K(m)) for xanthine oxidase was 13.51 μM and I(max) 0.071 μA. The biosensor measured xanthine in fish meat and lost 40% of its initial activity after its 200 uses over 100 days, when stored at 4°C.     

Identification of Hypoxanthine Transport and Xanthine Oxidase Activity in Brain Capillaries

Microvessel segments were isolated from rat brain and used for studies of hypoxanthine transport and metabolism. Compared to an homogenate of cerebral cortex, the isolated microvessels were 3.7-fold enriched in xanthine oxidase. Incubation of the isolated microvessels with labeled hypoxanthine resulted in its rapid uptake followed by the slower accumulation of hypoxanthine metabolites including xanthine and uric acid. The intracellular accumulation of these metabolites was inhibited by the xanthine oxidase inhibitor allopurinol. Hypoxanthine transport into isolated capillaries was inhibited by adenine but not by representative pyrimidines or nucleosides. Similar results were obtained when blood to brain transport of hypoxanthine in vivo was measured using the intracarotid bolus injection technique. Thus, hypoxanthine is transported into brain capillaries by a transport system shared with adenine. Once inside the cell, hypoxanthine can be metabolized to xanthine and uric acid by xanthine oxidase. Since this reaction leads to the release of oxygen radicals, it is suggested that brain capillaries may be susceptible to free radical mediated damage. This would be most likely to occur in conditions where the brain hypoxanthine concentration is increased as following ischemia.     

Biosensors for determination of total and natural antioxidant capacity of red and white wines: comparison with other spectrophotometric and fluorimetric methods

Research was carried out to experimentally evaluate the antioxidant capacity of several red and white wines using a superoxide dismutase (SOD) biosensor recently developed by the present authors. Measurements were performed by comparing the biosensor response to increasing concentration of the superoxide radical produced in solution by the xanthine/xanthine oxidase system, both in the presence and absence of the test sample.The results were compared with those of two traditional spectrophotometric methods and of a spectrofluorimetric method described in literature.Lastly, also the polyphenol, sulfite and ascorbic acid contents of the different wine samples examined were measured using a tyrosinase biosensor, a sulfite oxidase biosensor and an ascorbate oxidase biosensor, respectively.     

Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O ₂ ^⋅ and H₂O₂ during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-β-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O₂ consumption, only slightly but substantially inhibited in a competitive manner the O ₂ ^⋅ -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive Superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria. These results are interpreted in terms of a possible lysosomal membrane permeability to O ₂ ^⋅ causing organelle impairment by a process that, though leading to enzyme-marker leakage, does not involve lipid peroxidation.     

High-performance liquid chromatographic determination of hypoxanthine and xanthine in biological fluids

A rapid and selective reversed-phase high-performance liquid chromatographic method for the simultaneous determination of hypoxanthine and xanthine in biological fluids was developed. The identification of hypoxanthine and xanthine was confirmed by xanthine oxidase reaction. This method was applied to the investigation of purine metabolism in subjects with xanthine oxidase deficiency or gout. Hypoxanthine concentrations three to ten times higher than those determined in plasma were found in erythrocyte samples from normal subjects and from patients with xanthine oxidase deficiency or hyperuricemia under allopurinol therapy.     

Lack of conversion of xanthine dehydrogenase to xanthine oxidase during warm renal ischemia

Irreversible transformation of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) during ischemia was determined measuring XDH and total enzyme activity in kidneys before and after 60 min of clamp of the renal pedicle. Tissue levels of adenine nucleotides, xanthine and hypoxanthine were used as indicators of ischemia. After 60 min of clamping, ATP levels decreased by 72% with respect to controls whereas xanthine and hypoxanthine progressively reached tissue concentrations of 732 +/- 49 and 979 +/- 15 nmol.g tissue-1, respectively. Both total and XDH activities in ischemic kidneys (30 +/- 15 and 19 +/- 1 nmol.min-1.g tissue-1) were significantly lower than in controls when expressed on a tissue weight basis. The fraction of enzyme in the XDH form was however unchanged indicating that the reduction of the nucleotide pool is not accompanied by induction of the type-O activity of xanthine oxidase.     

Immobilization of a trienzymatic system in a sol-gel matrix: a new fluorescent biosensor for xanthine

In this work we report the development of a highly sensitive fluorescent multienzymatic biosensor for quantitative xanthine detection. This biosensor is built by the simultaneous encapsulation of three enzymes, xanthine oxidase, superoxide dismutase and peroxidase, in a single sol-gel matrix coupled to the Amplex Red probe. The sol-gel chemistry yields a porous, optically transparent matrix that retains the natural conformation and the reactivity of the three co-immobilized proteins. Xanthine determination is based on a sequence of reactions, namely catalytic oxidation of xanthine to uric acid and superoxide radical, and subsequent catalytic dismutation of the radical, resulting in the formation of hydrogen peroxide, which reacts stoichiometrically with non-fluorescent Amplex Red to produce highly fluorescent resorufin. The optimal operational conditions for the biosensor were investigated. Linearity was observed for xanthine concentrations up to 3.5 microM, with a detection limit of 20 nM, which largely improved the sensitivity of the current xanthine biosensors. The developed biosensor is reusable and remains stable for 2 weeks under adequate storage conditions.     

The pathophysiology of superoxide: roles in inflammation and ischemia

The superoxide radical plays major roles in the neutrophil-medicated acute inflammatory response and in postischemic tissue injury, although the sources and actions of the radical are quite different in these two pathological states. While neutrophils produce superoxide for the primary purpose of aiding in the killing of ingested microbes, a second useful function has evolved. The superoxide released from actively phagocytosing neutrophils serves to attract more neutrophils by reacting with, and activating, a latent chemotactic factor present in plasma. Superoxide dismutase, by preventing the activation of this superoxide-dependent chemotactic factor, exerts potent anti-inflammatory action. During ischemia, energy-starved tissues catabolize ATP to hypoxanthine. Calcium transients in these cells appear to activate a calmodulin regulated protease which attacks the enzyme xanthine dehydrogenase, converting it to a xanthine oxidase capable of superoxide generation. When the tissue is reperfused and reoxygenated, all the necessary components are present (xanthine oxidase, hypoxanthine, and oxygen) to produce a burst of superoxide which results in extensive tissue damage. Ischemic tissues are protected by superoxide dismutase or allupurinol, an inhibitor of xanthine oxidase.     

Methylprednisolone Attenuates Airway and Vascular Responses Induced by Reactive Oxygen Species in Isolated, Plasma-Perfused Rat Lungs

The effects of methylprednisolone (MP) on the acute airway and pulmonary vascular responses induced by reactive oxygen species (ROS) were investigated in isolated, plasma-perfused rat lungs. ROS were generated by adding xanthine oxidase and hypoxan-thine to the perfusate. MP was administered in 3 different ways: 1. Added to the perfusate (1 mg*ml^-1) 5 min prior to xanthine oxidase and hypoxanthine, 2. Given as intraperitoneal injections (40 mg*kg^-1) to lung donor rats 12 and 2 hours prior to the experiments, or 3. Combining 1 and 2. The lungs were perfused at constant volume inflow (15 ml*min^-1). Pulmonary arterial pressure and transpulmonary pressure were followed for 30 min after addition of xanthine oxidase and hypoxanthine. ROS induced a powerful, acute broncho- and vasoconstriction, which was inhibited by addition of MP to the perfusate. Pretreatment with MP also inhibited the vascular and airway responses. Adding MP to the perfusate of pretreated lungs further reduced the ROS-induced smooth muscle constriction. In conclusion, MP inhibits vasoconstriction and bronchocon-striction induced by ROS in isolated rat lungs.