Immobilized enzymatic fluorescence capillary biosensor for determination of sulfated bile acid in urine

The authors have proposed an immobilized enzymatic fluorescence capillary biosensor (SBAs-IE-FCBS) for the determination of sulfated bile acids (SBAs). The reaction principle of the biosensor is that under the catalysis of the bile acid sulfate sulfatase (BSS) and beta-hydroxysteroid dehydrogenase (beta-HSD) immobilized on inner surface of a medical capillary, SBAs desulfates to 3 beta-hydroxyl bile acids, then the latter reacts with nicotinamide adenine dinucleotide (NAD(+)), and is converted into 3-ketosteroid; meanwhile, NAD(+) is converted to reduced nicotinamide adenine dinucleotide (NADH). NADH continuously reacts with 1-methoxy-5-methylphenazinium methyl sulfate (1-MPMS) and is converted into NAD(+) circularly and 1-MPMSH2. Finally resazurin is reduced into resorufin by 1-MPMSH2, the formed resorufin (lambda(ex)/lambda(em) : 540 nm/580 nm) is used for quantifying the concentration of SBAs. Optimized conditions being suitable with the biosensor are as follows: the concentrations of BSS and beta-HSD used for the immobilization all are 5 kU L-1; the concentrations of 1-MPMS and resazurin all are 25 mu mol L-1; the concentrations of Tris-HCl buffer and NAD(+) are 100 and 400 mu mol L-1, respectively; total volume of the enzyme, reagent and sample is only 18 mu L per time for determining; the reaction temperature is 37 degrees C; the reaction time is 15 min. The concentration of SBAs is directly proportional to the fluorescence intensity of the biosensor measured from 0.5 to 5.0 mu mol L-1. The relative standard deviation is less than 3.4%, and the detection limit was 0.16 mu mol L-1. The recoveries are in the range 95.5-106%. This SBA-IE-FCBS can be used for quantifying SEAs in urine to diagnose and judge hepatobiliary diseases, etc. (C) 2008 Elsevier B.V. All rights reserved.