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 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.
Li
Yong-Sheng
YS
School of Chemical Engineering, Sichuan University, Chengdu 610065, China.
Liu
Wei-Ping
WP
Gao
Xiu-Feng
XF
Chen
Dan-Dan
DD
Li
Wei-Guang
WG
D001647:Bile Acids and Salts / Q000652:urine*; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D004800:Enzymes, Immobilized / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods; D013429:Sulfatases / Q000737:chemistry*; D013431:Sulfates / Q000652:urine*; D016482:Urinalysis / Q000295:instrumentation* / Q000379:methods
D001647:Bile Acids and Salts; D004800:Enzymes, Immobilized; D013431:Sulfates; D006913:Hydroxysteroid Dehydrogenases; D013429:Sulfatases; C033345:bile salt sulfatase