Xiu-Feng Gao

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.

Determination of pyruvic acid by using enzymic fluorescence capillary analysis

A new method (P-LE-FCA) for the determination of pyruvic acid was proposed based on liquid enzyme method (LE) and fluorescence capillary analysis (FCA). The optimum experimental conditions were as follows: the excitation and emission wavelengths were 350 and 460 nm, respectively; the reaction time and temperature were 20 min and 38 degrees C, respectively; the pH of phosphate buffer solution was 7.5; the concentrations of nicotinamide adenine dinucleotide and lactate dehydrogenase were 1.0 mmol L(-1) and 5.0 k UL(-1), respectively. The linear range of this method was 0.2-1.2 mmol L(-1) (Delta F=327.13C-10.018, r=0.9942). Its detection limit was 0.012 mmol L(-1). And its relative standard deviation was 0.86%. Only 18 microL of total reaction solution is enough for the detection. P-LE-FCA has some merits such as lower cost, simple operation procedure and micro determination. It has been used for the determination of pyruvic acid content in human urine samples.

A novel immobilization multienzyme glucose fluorescence capillary biosensor

Based on the immobilization enzyme technology and the fluorescence capillary analysis method, the authors have developed a highly sensitive fluorescence reaction system and a novel immobilization multienzyme glucose fluorescence capillary biosensor for determining glucose contents. Reaction principle of the system is that under the catalysis of glucose oxidase (GOD) and horseradish peroxidase (HRP) immobilized on inner surface of a medical capillary, beta-D-glucose reacts with dissolved oxygen to form gluconic acid-delta-lactone and hydrogen peroxide, and then the latter reacts with l-tyrosine to produce a tyrosine dimer, which has maximal excitation and emission wavelengths at 320 nm and 410 nm, respectively. Fluorescence of the dimer is proportional to the concentration of the beta-D-glucose. Optimization conditions suitable for the reaction system and the biosensor were as follows. Concentration of the L-tyrosine used as fluorescence reagent was 0.15 mol L(-1), the active concentrations of the GOD and the HRP for the immobilization were 15 kU L(-1) and 8 kU L(-1), respectively. Consumptions of the sample and reagents in one determination were 5.0 microL and 15 microL, respectively. Quantitative range of the biosensor for the glucose was in the range 1-10 micromol L(-1), its relative standard deviation was less than 4.9%, and its detection limit was 0.62 micromol L(-1). The biosensors recovery was in the range 96-105%. Results of some serum determined with the biosensor and with a commercial glucose-kit were well coincident to each other. Accordingly, the biosensor can be applied to the determination of serum glucose contents in the diagnosis of diabetes.

Immobilization enzyme fluorescence capillary analysis for determination of lactic acid.

A new method for the determination of lactic acid based on the immobilization enzyme fluorescence capillary analysis (IE-FCA) was proposed. Lactic dehydrogenase (LDH) was immobilized on inner surface of a capillary with glutaraldehyde, and an immobilized enzyme lactate capillary bioreactor (IE-LCBR) was formed for the determination of lactic acid. After nicotinamide adenine dinucleotide (NAD(+)) is mixed with lactic acid solution, it was sucked into the IE-LCBR and was detected at lambda(ex) 353 nm/lambda(em) 466 nm. Optimized conditions are as follows: the temperature is 38 degrees C; the reaction time is 15 min; the concentrations of Tris buffer (pH 8.8) and NAD(+) are 0.1 mol L(-1) and 4 mmol L(-1), respectively; the concentration of LDH used for immobilization is 15 kU L(-1). The concentration of lactic acid is directly proportional to the fluorescence intensity measured from 0.50 to 2.0 mmol L(-1); and the analytical recovery of added lactic acid was 99-105%. The minimum detection limit of the method is 0.40 mmol L(-1) and sensitivity of the IE-CBR is 4.6 F mmol(-1) L(-1) lactate. Its relative standard deviation (R.S.D.) is < or =2.0%. This IE-FCA method was employed for determination of lactate in milk drink.

A novel assay for determination of sulfated bile acids in urine by use of flow-injection chemiluminescence principle with immobilized enzymes

Multistep enzymatic reactions take place when sulfated bile acids (SBAs) pass through an immobilized enzyme reactor. First, SBAs take place in the reaction of desulfation under catalytical action of a bile salt sulfatase immobilized in the reactor and formed 3β-hydroxyl bile acids. Formed 3β-hydroxyl bile acids react with nicotinamide adenine dinucleotide (β-NAD+) under catalysis of another enzyme of 3β-hydroxysteroid dehydrogenase coimmobilized in the column and are converted to 3-ketosteroid. At the same time, β-NAD+ is changed into reduced nicotinamide adenine dinucleotide (NADH). Looking as if chain reaction, 1-MPMS taken as an electron mediator reacts immediately with NADH coexisting in the carrier solution and is turned into 1-MPMSH2. Formed 1-MPMSH2 again reacts with dissolved oxygen existing in the carrier solution and produces hydrogen peroxide. Last, the hydrogen peroxide reacts with the luminol reagent and gives out light in the presence of POD. Consequently, SBA can be determined by the luminous intensity. Based on the above-mentioned principle, applying the technology of flow-injection analysis, we established an accurate, simple, less time-consuming and sensitive approach for the determination of SBAs. According to the FIA manifold and experimental results optimized in the work, a new type of analytical instrument for the clinical assay of SBAs in urine or blood can be developed. We also think this method is very useful for routine analysis in clinical laboratory and long-period monitoring to patients with acute hepatitis, liver cirrhosis, and intra- and extrahepatic biliary obstruction of the urinary tract. Sampling frequency of the method is 30 samples/h, and its relative standard deviation is smaller than 2.2%. It can be used to determine SBAs in the ranges of 0.1–2.5 or 2–25 μM.

Heterologous expression and purification of aldehyde dehydrogenase gene from Bacillus halodurans XJU-1.

BACKGROUND For its function of catalysing the reaction of aldehyde to acetic acid, ALDH could be applied to antialcoholismic drug development. However, both of the production and activity of natural ALDH are too low to meet the demand. OBJECTIVE To empirically explore whether aldh gene in Bacillus halodurans XJU-1 could be highly expressed in E.coli BL21(DE3) and to investigate the purification of recombinant protein ALDH. METHODS The aldh gene in Bacillus halodurans XJU-1 was cloned into prokaryotic expression vector pET30b(+), then transformed into E.coli BL21(DE3) to induce expression of ALDH. Immobilized metal ion affinity chromatography (IMAC) was applied in the separation and purification for ALDH proteins. RESULTS The recombinant vector for aldh gene was constructed and expressed in BL21(DE3) successfully. The ALDH recombinant protein was purified by ammonium sulfate precipitation and IMAC, and obtained 30.1% of the recovery with a purification factor 8.81. CONCLUSIONS The value of these findings, as well as wider implications for increasing the yield and the activity of ALDH, is meaningful.