An Amperometric Oxalate Biosensor Based on Sorghum Leaf Oxalate Oxidase Immobilized on Carbon Paste Electrode

Abstract

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 <7.5% and <8.5% in urine of healthy persons and <5.0% and <6.5% in the urine of stone formers, respectively. There was a good correlation (r = 0.91) between urinary oxalate values by present method and the most commonly used enzymatic colorimetric method. The enzyme electrode was used for 150-times during the span of 35 days, when stored in a reaction buffer at 4°C.

Keywords:

• Amperometric biosensor
• Beer
• Carbon paste electrode
• Grain sorghum
• Oxalate
• oxalate biosensor
• Oxalate oxidase
• Urine

Acknowledgments

The authors are thankful to Ms/s Nath Seeds Pvt. Ltd, Aurangabad, India for providing the seeds of grain sorghum (var. Amarnath), and Prof. R. S. Chaudhary, Department of Chemistry, M. D. U. Rohtak and his ex. Ph.D student Dr. Harish and Mr. Vikas, Department of BioSciences, M. D. U. Rohtak for their help in using the potentiostat and the preparation of the carbon paste electrode, respectively.

Notes

Six samples of each were taken and mean was calculated.

Mean value of oxalate in male was 11.6 µg/ml.

Mean value of oxalate in female was 6.6 µg/ml.

Mean value of oxalate in male was 20.0 µg/ml.

Mean value of oxalate in female was 19.3 µg/ml.

Mean value of oxalate in beer was 11.8 µg/ml.