Novel determination system for urea in alcoholic beverages by using an FIA system with an acid urease column

A novel determination method for urea using an acid urease column-FIA system was developed, and the system was applied to the determination of urea in rice wine. This novel FIA system was characterized by CO2 detection due to the property of acid urease and by a microfluidic gas-diffusion device with the use of an ultra-thin hollow fiber membrane. A biosensing system fabricated in this study was assembled with a double-plunger pump, a sample-injection valve, an immobilized acid urease column as a recognition element for the assay of urea, a gas-diffusion unit, and a flow-type spectrophotometer. The gas-diffusion unit consisted of a double-tubing structure in which the outer tubing was made of PTFE (i.d. 1.0 mm; o.d. 1.5 mm) and the inner tubing was of porous PTFE (i.d. 0.19 mm; o.d. 0.25 mm). Standard urea solutions (20 microl) were measured through monitoring variations in the absorbance of a coloring agent solution resulting from a pH shift due to carbon dioxide molecules being enzymatically generated. A wide and linear relationship was obtained between the concentration of urea (16 microM - 1.0 mM) and the change in absorbance. This FIA system has great advantages that the system did not suffer from ammonia and ethanol in samples. This system, armed with a microfluidic gas-diffusion device, was applicable to the determination of various substrates of many kinds of decarboxylase, amino-acid oxidase, and amino-acid oxygenase, producing CO2 and NH3 molecules.