Fabrication of dissolved O2 metric uric acid biosensor using uricase epoxy resin biocomposite membrane

Anal Chim Acta. 2009 Aug 11;647(2):195-201. doi: 10.1016/j.aca.2009.06.001. Epub 2009 Jun 7.

Abstract

Uricase purified from 20-day-old leaves of cowpea was immobilized on to epoxy resin membrane with 80% retention of initial activity of free enzyme and a conjugation yield of 0.056 mg/cm(2). The uricase epoxy resin bioconjugate membrane was mounted over the sensing part of the combined electrode of 'Aqualytic' dissolved O(2) (DO) meter to construct a uric acid biosensor. The biosensor measures the depletion of dissolved O(2) during the oxidation of uric acid by immobilized uricase, which is directly proportional to uric acid concentration. The biosensor showed optimum response within 10-12s at a pH 8.5 and 35 degrees C. A linear relationship was found between uric acid concentration from 0.025 to 0.1 mM and O(2) (mg/l) consumed. The biosensor was employed for measurement of uric acid in serum. The mean value of uric acid in serum was 4.92 mg/dl in apparently healthy males and 3.11 mg/dl in apparently healthy females. The mean analytic recoveries of added uric acid in reaction mixture (8.9 and 9.8 mg/dl) were 93.6 +/- 2.34 and 87.18 +/- 3.17% respectively. The within and between batch CVs were < 6.5 and < 5.0%, respectively. The serum uric acid values obtained by present method and standard enzymic colorimetric method, showed a good correlation (r - 0.996) and regression equation being y - 0.984x + 0.0674. Among the various metabolites tested only, glucose (11%), urea (38%), NaCl (25%) and cholesterol (13%) and ascorbic acid (56%) caused decrease, while, MgSO(4) and CaCl(2) had no effect on immobilized enzyme. The enzyme electrode showed only 32% decrease during its use for 100 times over a period of 60 days at 4 degrees C.

Publication types

  • Validation Study

MeSH terms

  • Biosensing Techniques / instrumentation*
  • Biosensing Techniques / methods*
  • Enzymes, Immobilized / chemistry
  • Enzymes, Immobilized / isolation & purification
  • Enzymes, Immobilized / metabolism
  • Epoxy Resins / chemistry
  • Equipment Design
  • Fabaceae / chemistry*
  • Female
  • Humans
  • Kinetics
  • Male
  • Membranes, Artificial
  • Microscopy, Electron, Scanning
  • Oxidation-Reduction
  • Oxygen / analysis
  • Plant Leaves / chemistry*
  • Sensitivity and Specificity
  • Urate Oxidase / chemistry
  • Urate Oxidase / isolation & purification
  • Urate Oxidase / metabolism*
  • Uric Acid / blood*

Substances

  • Enzymes, Immobilized
  • Epoxy Resins
  • Membranes, Artificial
  • Uric Acid
  • Urate Oxidase
  • Oxygen