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

Biosens Bioelectron. 2008 Dec 1;24(4):538-44. doi: 10.1016/j.bios.2008.05.001. Epub 2008 May 27.


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 3beta-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 (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 kUL(-1); the concentrations of 1-MPMS and resazurin all are 25 micromolL(-1); the concentrations of Tris-HCl buffer and NAD(+) are 100 and 400 micromolL(-1), respectively; total volume of the enzyme, reagent and sample is only 18 microL per time for determining; the reaction temperature is 37 degrees C; the reaction time is 15min. The concentration of SBAs is directly proportional to the fluorescence intensity of the biosensor measured from 0.5 to 5.0 micromolL(-1). The relative standard deviation is less than 3.4%, and the detection limit was 0.16 micromolL(-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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bile Acids and Salts / urine*
  • Biosensing Techniques / instrumentation*
  • Biosensing Techniques / methods
  • Enzymes, Immobilized / chemistry
  • Equipment Design
  • Equipment Failure Analysis
  • Hydroxysteroid Dehydrogenases / chemistry*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Spectrometry, Fluorescence / instrumentation*
  • Spectrometry, Fluorescence / methods
  • Sulfatases / chemistry*
  • Sulfates / urine*
  • Urinalysis / instrumentation*
  • Urinalysis / methods


  • Bile Acids and Salts
  • Enzymes, Immobilized
  • Sulfates
  • Hydroxysteroid Dehydrogenases
  • Sulfatases
  • bile salt sulfatase