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

Abstract

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 β-hydroxysteroid dehydrogenase (β-HSD) immobilized on inner surface of a medical capillary, SBAs desulfates to 3β-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-MPMSH 2. Finally resazurin is reduced into resorufin by 1-MPMSH 2, the formed resorufin ( λ ex/ λ 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 β-HSD used for the immobilization all are 5 kU L −1; the concentrations of 1-MPMS and resazurin all are 25 μmol L −1; the concentrations of Tris–HCl buffer and NAD + are 100 and 400 μmol L −1, respectively; total volume of the enzyme, reagent and sample is only 18 μL per time for determining; the reaction temperature is 37 °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 μmol L −1. The relative standard deviation is less than 3.4%, and the detection limit was 0.16 μmol L −1. The recoveries are in the range 95.5–106%. This SBA-IE-FCBS can be used for quantifying SBAs in urine to diagnose and judge hepatobiliary diseases, etc.