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An amperometric oxalate biosensor was developed and optimized by immobilizing a Cl− and insensitive oxalate oxidase purified from sorghum leaves, on carbon paste electrode. Enzymatically produced H2O2 from oxalate was split into 2H+ + O2 + 2e− at 60 mV and flow of electron (current) was measured. The biosensor showed optimum response at pH 4.5, 35°C and within 45 sec. The current measured (mA) by biosensor was linear for oxalate up to 20 µg/ml. The biosensor was employed for determination of oxalate in urine (from both apparently healthy individuals and urinary stone formers) and different brands of beer. The percent recoveries of added oxalate in urine of healthy and stone formers were 96.6% and 98.2%, respectively. Within and between batch coefficients of variation (CV) for oxalate determination were

A method has been developed for discrete analysis of urinary oxalate employing alkylamine glass bound oxalate oxidase partially purified from mature leaves of Amaranthus spinosus. In the method H2O2 is generated from urinary oxalate by immobilized oxalate oxidase, which is measured by a colour reagent containing 4-aminophenazone, phenol and horseradish peroxidase in phosphate buffer, pH 7.0. The chromogen formed is read at 520nm. The minimum detection limit of the method is 0.9mg/L. The analytical recovery of added oxalate in urine was 98.0±2.7% (mean±SD). Within batch and between batch CV's were less than 4% and 3%, respectively, for urinary oxalate determination. The mean value of urinary oxalate measured by the present method is comparable to that obtained by the Sigma kit method (r=0.98).

Oxalate oxidase, isolated from banana fruit peel, is immobilized on various supports and the efficacies of these derivatives in determining urinary oxalate are compared. Banana oxalate oxidase, when complexed with its antiserum, is found to have higher specific activity, greater stability, broader range of pH profile and higher resistance to thermal inactivation. Oxalate oxidase, when immobilized in acrylamide membrane strip, is found to be stable and easy to use in the determination of urinary oxalate. These two preparations compare well with that of soluble enzyme in sensitivity, accuracy and reliability. However, both hydrazide bead-linked oxalate oxidase and ethylene maleic anhydride derivative of oxalate oxidase are found to be unsuitable for the determination of urinary oxalate.

A flow injection analysis (FIA) method for the assay of oxalate in urine samples is described, which utilizes an incorporated column reactor of immobilized oxalate oxidase. The hydrogen peroxide generated is detected by chemiluminescence via reaction with luminol and hexacyanoferrate(III). Special empasis is placed on the role of L-ascorbic acid, which has been claimed to constitute the most serious interference in these types of assays. The possible elimination af this constituent by addition of sodium nitrite, as previously used in batch procedures, did not improve the selectivity of the approach. The FIA approach described, which has a detection limit (3[sgrave]) of 34 μM in undiluted urine samples and allows a sampling frequency of ca. 55 s/h, is shown to offer itself as convenient and rapid means for screening purposes.

Oxalate oxidase (oxalate:oxygen oxidoreauctase, FC 1.2.3.4), isolated from ripe banana peel was precipitated by rat antiserum. Compared with native enzyme, this immune complex possessed higher specific activity, greater stability, broader pH range for optimal activity and higher resistance to thermal and heavy metal inactivation. These improved characteristics of the immune complex make it ideally suited for its use in the determination of urinary oxalate. A simple method of determining oxalate in urine samples, using this preparation is described.

We describe in this paper a simple and easy method of entrapping oxalate oxidase and peroxidase in acrylamide membrane and demonstrate the use of such enzyme membrane strips in the rapid determination of urinary oxalate. A crude preparation of 45-60% acetone cut obtained from banana fruit peel (Musa paradisiaca; French plantain) homogenate serves as a source of oxalate oxidase, which decomposes oxalate into CO2 and H2O2. the oxalate content of a given urine sample is determined by introducing a small enzyme membrane strip (1 × 1 cm) into an aliquot of buffered urine containing a suitable chromogen for peroxidase and then measuring the colour developed due to the interaction of peroxidase with the newly formed H2O2 and the chromogen. Urine samples are pretreated with sodium nitrite to eliminate the interference of ascorbic acid in the assay. the enzyme membrane assay compares well with that of wet enzyme assay of oxalate in sensitivity and reliability.

An enzyme membrane electrode usable for the assay of oxalate in foodstuffs is described. A commercially available preactivated polyamide membrane was used for the immobilization of oxalate oxidase. The bioactive disk thus obtained was associated with an amperometric transducer. The resulting self-contained enzyme electrode wich allows oxalate determination in various materials with minimal pretreatment exhibits a linear calibration ranging from 10–7 M and 10–4 M in the cell. The response-time was comprised between 20 seconds and 1 minute, depending on the oxalate content in the sample. The electrode-response was very stable for at least 4 months, a period during which more than 150 assays were performed. The results obtained with several food materials were in good agreement with those obtained with the conventional spectrophotometric method. Assays were also performed with a microprocessor-based analyzer normally used for glucose measurements with a glucose oxidase electrode When the analyzer is...

This paper describes a rapid enzymic procedure, based on calorimetry, for specific determination of oxalic acid. Oxalate is oxidized by immobilized oxalate oxidase to hydrogen peroxide and carbon dioxide. The heat generated by this reaction is measured in a calorimetric device, the enzyme thermistor. Oxalate concentrations as low as 0.02 mM can be determined. Also described is purification and immobilization of the enzyme, as well as the effect of some of its inhibitors. The urinary oxalate content of 16 persons was determined using the enzyme thermistor. For samples containing a very low concentration of oxalate (less than 0.2 mM) an extraction step with tributylphosphate can be introduced to purify and concentrate the oxalate. The oxalate content in different kinds of food was also determined.

Enzyme electrodes for oxalate were constructed by immobilizing oxalate oxidase enzyme (E, C, 1,2,3,4) on both potentiometric carbon dioxide and amperometric hydrogen peroxide sensors. These systems were optimized, and the response characteristics of the two biosensors examined in a comparison study. Areas investigated include linear range, calibration curve slope, response time, limit of detection, and lifetime. Selectivity studies were carried out with the system and showed no measurable response to millimolar levels of various L-amino acids, metal ions or ascorbic acid. This comparison was extended into the choice of an optimal sensor for urinary oxalate determinations with minimal sample pretreatment. The peroxide sensor performed best in urine samples, allowing for the 1:40 dilution necessary to minimize the effect of any interferents present. At this or greater dilution, the recovery of standard additions of oxalate averaged 95.5%, with an average relative standard deviation of 4.9%. The sys...

Carboxyl groups on a collagen membrane were activated with the aid of an acyl-azide reaction. the enzyme oxalate oxidase, prepared from barley seedlings, was subsequently immobilized on the activated collagen film. the specific activity and the Michaelis constant were 0.13 units/cm2 and 0.1 M, respectively. Enzyme activities were determined as function of pH, ionic strength, and ambient chloride concentration, intercomparing the enzyme in aqueous solution with its immobilized counterpart. Oxalate was quantitated amperometrically via electrooxidation of the hydrogen peroxide by-product generated in the reaction between oxalic acid and O2. the detection limit of oxalate was 10−5, which yielded a current of 2 nanoampere.

This report describes the construction and characterization of an oxalate-sensing electrode. The electrode is based on the incorporation of ground beet stem into the graphite paste of a graphite paste electrode. The hydrogen peroxide generated by enzymatic degradation of oxalate is monitored at a working voltage of 0.900 V vs SCE. All measurements were conducted in a succinic acid/EDTA buffer at pH 4.00. Under these conditions, the electrodes exhibit reproducible responses to oxalate. The lower limit of oxalate detection was less than 1.03 × 10−4 M. The time to achieve a steady state response after exposure to a step change in oxalate concentration in solution is less than one minute. The magnitude of response to oxalate over the oxalate concentrations studied varies among several electrode tested as does the degree of linearity of response. An electrode studied still exhibited analytically useful responses to oxalate on the 15th day of its use. The beet stem-based electrodes display little respo...