Development of a micro-planar amperometric bile acid biosensor for urinalysis.

The determination of bile acid concentration in urine is useful for the screening and diagnosis of various hepatobiliary diseases. Currently, there is no concise method to determine bile acid concentration in urine. This study describes a bile acid biosensor fabricated by electrochemical technique for urinalysis. The micro-planar electrodes employed for the study consisted of a working electrode (platinum), a counter electrode (platinum) and a reference electrode (silver/silver chloride (Ag/AgCl)). The sensor chip was coated with Nafion using a spin-coater in order to both eliminate many interference species in urine and achieve long-term stability of the reference electrode. Nafion coating allowed the sensor chip to prevent the electrode reaction from interference species in urine, because it is charged negative strongly (Nafion contains sulfonic acid group). Three enzymes (bile acid sulfate sulfatase: BSS, beta-hydroxysteroid dehydrogenase: beta-HSD, and NADH oxidase: NHO) were immobilized by glutaraldehyde (GA: cross-linker) onto the sensor chip, because the immobilization of enzymes by GA is simple and commonly carried out. The sensor chip was able to detect bile acid in buffer solution. The optimum enzyme ratio immobilized onto the sensor chip was BSS:beta-HSD:NHO=4:4:20 U/1 chip. There was a relationship between the concentration of bile acid and the response current value. The dynamic range of the sensor chip was 2-100 microM for bile acid. Additionally, bile acid in the urine specimen could be detected using this bile acid biosensor. We present a simple and rapid bile acid biosensor with high sensitivity and high reproducibility.

Interaction of three-way DNA junctions with steroids.

DNA aptamers that bind to cholic acid were previously isolated by an in vitro selection method. Secondary structural prediction and deletion-mutant experiments suggested that the cholic-acid binding regions of 19 sequenced clones could form three-way-junction structures. In this article, the secondary structures of the sequenced clones and the structural requirements for binding to cholic acid were evaluated. A course of mutational-analysis and chemical-modification experiments provided strong support for the predicted secondary structure and also indicated that the binding site is located at the branching point of the three-way junction. Sequence analysis revealed that the sequences of the three base pairs flanking the junction of the three stems are highly conserved among selected clones. The evaluation of the relative binding of several bile acids and structurally related steroids with the aptamer was also carried out. The results revealed a broad range of selectivity and preference for hydrophobic steroids rather than for cholic acid upon binding, indicating that the binding is driven by a hydrophobic interaction. The experimental results reported here allowed us to propose a structural model of a binding site formed by three Watson-Crick base pairs.

Effect of alcohols on the collagen-phosphatidylcholine interaction.

The interaction of phosphatidylcholine dispersions with acid soluble collagen separated from the skin of one month-old swine was studied to define the conditions facilitating the association of the collagen with lipids. When acid soluble collagen and phosphatidylcholine dispersions were incubated in 75 mM citrate buffer of pH 3.7 at 25 degrees C, the reisolated collagen fibrils did not contain appreciable amounts of phosphatidylcholine. However, the presence of n-propanol greatly promoted the retention of phosphatidylcholine, the amount of phosphatidylcholine associated being nearly 30% of collagen on a weight basis under optimal conditions. In contrast, methanol, ethanol, isopropanol, and n-butanol did not appreciably enhance the association of phosphatidylcholine with collagen. A limited inhibition of phosphatidylcholine retention was observed upon addition of sodium chloride to the propanol medium. The interaction of phosphatidylcholine with acid soluble collagen decreased sharply when temperature was increased above 30 degrees C; almost no phosphatidylcholine-collagen association occured at 40 degrees C. It appears that the enhanced association in the presence of n-propanol is due to a looseing of the collagen triple helix that exposes hydrophobic sites necessary for the interaction. However, the conversion of the triple helical structure to the random coil conformation by heating prevents the association of phosphatidylcholine with acid soluble collagen.

Photocontrol of urease activity in spiropyran collagen membrane.

1. Collagen fibrils were modified with beta-1-[3,3-dimethyl-6'-nitrospiro-(indoline-2,2'-2H-benzopyran)] propionic anhydride. 2. Urease (urea amidohydrolase, EC 3.5.1.5) was immobilized in spiropyran collagen membrane. The activity of the urease-spiropyran collagen membrane was found to increase in the dark and then decrease with visible light irradiation. 3. The optimum pH of the urease-spiropyran collagen membrane under visible light was lowered in the dark. 4. The apparent Michaelis constant (K'm) of the urease-spiropyran collagen membrane in the dark was almost the same as that under visible light. The apparent maximum velocity was increased in the dark. 5. The diffusion coefficient of urea through the spiropyran collagen membrane in the dark was 1.4 times that under visible light. However, the increase of the diffusion rate was not responsible for the activity increase of the urease-spiropyran collagen membrane.

NAD recycling in the collagen membrane.

NAD recycling in the collagen membrane was investigated as follows: (1) Alcohol dehydrogenase and lactate dehydrogenase were co-immobilized in the collagen membrane and the rate of lactate production by immobilized enzymes was compared with that of free enzymes by using free NAD. An increased rate was observed in the case of immobilized enzyme. (2) The soluble high molecular weight derivatives of NAD (dextran-NAD) were immobilized in the collagen membrane with the two dehydrogenases and recycling of dextran-NAD in the membrane was examined. Lactate was produced by the membrane without adding free NAD. The interaction between the high molecular weight NAD derivatives and enzymes are also discussed.

Photocontrol of urease-collagen membrane activity.

(1) Urease (EC 3.5.1.5.) was modified with beta-1-[3,3-dimethyl-6'-nitrospiro-(indoline-2,2'-2H-benzopyrene)] propionic anhydride. Three amino acid residues of urease were modified by the anhydride at a molar ratio of 2000. (2) The activity of modified urease was decreased with ultraviolet irradiation and then restored to the initial activity with visible light irradiation. (3) Modified urease was used to prepare a urease-collagen membrane. The apparent Michaelis constant (Km) of the modified urease-collagen membrane ultraviolet light was identical to that of the membrane under visible light. (4) The optimum pH of the modified urease-collagen membrane was displaced toward lower pH values with ultraviolet irradiation. At higher ionic strength, the pH activity curve of the membrane was displaced toward higher pH values. (5) The thermostability of urease was increased with its modification.

Investigation of the potential active site of a cyanide dihydratase using site-directed mutagenesis.

Cyanide dihydratase has conserved residues in the amino acid sequence with nitrilase, and cyanide hydratase. The conserved amino acid residues in the cyanide dihydratase from Pseudomonas stutzeri AK61 were altered by site-directed mutagenesis. The enzyme completely lost its activity of the cyanide hydrolysis by the replacement of cysteine-163 to serine. The replacement of tyrosine-53 to phenylalanine caused an increase of the K(m) value of the enzyme for cyanide. Substitution of nine other residues seemed to affect the structure of the enzyme.

Electrical stimulation of hybridoma cells producing monoclonal antibody to cAMP.

Electrical stimulation was applied to hybridoma cells in order to activate metabolic activities and increase the monoclonal antibody production. Hybridoma cells that produce monoclonal antibody to adenosine 3':5'-cyclic monophosphate were placed on a transparent glass electrode immersed in medium and subjected to electric pulses (pulse shape, alternating rectangular; field strength, 4 X 10(3) V X m-1; frequency, 5 kHz; pulse mode, 0.5 min application and 4.5 min pause). After 48 h of incubation, the concentration of lactic acid in the medium reached 8.4 mM, approx. 30% higher than that obtained without electric stimulation. Similarly, cell growth rate was promoted by the electric stimulation, reaching a maximum stimulation after 40 h. When the hybridoma was cultured for 48 h with electrical stimulation, the antibody concentration in the medium reached 22.3 microgram X ml-1, approx. 10% higher than the control, with a concomitant 16% increase in cell concentration. Longer periods of electric pulse application, however, caused an inhibitory effect on the hybridoma growth. The most probable cause of the inhibition are reactive oxygen species such as superoxide and hydrogen peroxide, which are inevitably generated by electrolysis. The presence of superoxide dismutase (EC 1.15.1.1) reduced the inhibitory effects. In conclusion, metabolic activities including monoclonal antibody production were activated by the electrical stimulation.

In vitro selection of DNA aptamers which bind to cholic acid.

DNA aptamers which bind to cholic acid have been identified by in vitro selection from a pool of approximately 9x10(14) DNA molecules. After 13 rounds of selection, 19 clones with 95-100 nucleotide length were sequenced. Deletion-mutant experiments and computational sequence analysis suggested that all clones contained cholic acid binding sequences which could fold into three-way junction structures. By comparing the sequences involved in the predicted three-way junction structure of these 19 clones, it was determined that the nucleotide sequences and lengths of three stem and loop regions have no similarity. The most conserved structure seems to have three base pairs flanking the junction of the three stems and they may form a hydrophobic cavity in which they interact with cholic acid.

Immobilized cells used for detection and analysis.

Many kinds of biosensor have now been developed and utilized for various types of analysis including clinical, medical and environmental monitoring, industrial process control, as well as many other applications. The microbial biosensor has advantages, such as longer lifetime and lower cost, over other types of biosensor. Recently, photobacteria and recombinant bacteria have been employed in biosensors both for determining biochemical oxygen demand and for detecting heavy metals and other toxic compounds.

Biosensors and flow injection analysis.

Combining flow injection analysis with a biosensor is a novel biosensing process which has allowed speedy and accurate analysis. Diagnostic analysis is the most important application for biosensing flow injection analysis, but other applications include bioprocess monitoring, analysis of food and agricultural products, as well as environmental analysis. In addition, the analysis of compounds, such as explosives and abused drugs, and monitoring of Salmonella, the microorganism that causes food poisoning, have been reported.

The development of microfabricated biocatalytic fuel cells.

The production of electricity by biocatalytic fuel cells has been feasible for almost two decades and can produce electric power at a practical level. These fuel cells use immobilized microorganisms or enzymes as catalysts, and glucose as a fuel. A microfabricated enzyme battery has recently been made that is designed to function as a power supply for microsurgery robots or artificial organs.

New cell fusion method using polymer membrane.

A porous polymer membrane of nitrocellulose or tetrafluoroethylene (TFE) was employed for fusion of Saccharomyces cerevisiae (AH22 and D13-1A) protoplasts. Protoplasts were adsorbed on the membrane with slight suction. Some part of the protoplasts was trapped in pores of the membrane as observed by electron microscopy. The membrane retaining protoplasts was placed on a selective medium. Several colonies appeared on the medium after 5-7 days incubation at 30 degrees C. The fusion of the two strains was ascertained by DNA content and genetic markers. Fusion frequency was 1.2 X 10(-6) in the case of the TFE membrane.

Cloning and expression of the hydrogenase gene from Clostridium butyricum in Escherichia coli.

Hydrogenase gene from Clostridium butyricum was cloned in Escherichia coli HK16 (Hyd-) using pBR322 and PstI. The plasmid, pCBH1, containing hydrogenase gene was 7.3 MDa and pCBH1 had 5 PstI-DNA fragments (3.9, 2.6, 0.7, 0.03-0.04, less than 0.02 MDa, respectively). The hydrogenase activity of HK16 (pCBH1) was about 3.1-3.5-times as high as those of the present strains, such as C.butyricum and E.coli C600 (Hyd+).

DNA cleavage based on high voltage electric pulse.

A high voltage electric pulse was applied to DNA cleavage. The DNA cleavage reaction was dependent on the voltage amplitude, pulse number and pulse width. Radical scavengers and ESR data indicated the possibility that active species such as OH radical were strongly related to DNA cleavage.

Microbial sensors for volatile compounds.

A microbial sensor consisting of immobilized microorganisms, a gas permeable membrane, and an oxygen electrode was prepared for the continuous determination of methyl alcohol. Immobilized methyl alcohol utilizing bacteria was employed for the sensor. The response time of the sensor was within 10 min by the steady-state method. A linear relationship was observed between the current decrease and the concentration of methyl alcohol below 22.5 mg l-1. The selectivity of the microbial sensor for methyl alcohol was satisfactory. Microbial sensors using Trichosporon brassicae for ethyl alcohol and acetic acid are also described. A microbial sensor consisting of immobilized nitrifying bacteria (isolated from activated sludges), a gas-permeable Teflon membrane and an oxygen electrode was prepared for the amperometric determination of ammonia. When the sensor was inserted in a solution containing ammonia, the current decreased to a steady-state with a response time of 4 min. The relationship between the current decrease and the ammonia concentration was linear up to 42 mg l-1. The minimum concentration for the determination was 3.5 mg l-1. The current decrease was reproducible within 4 per cent of relative error. The current output of the sensor was almost constant for over 10 days and 200 assays.

Biochemical energy conversion using immobilized whole cells of Clostridium butyricum.

Hydrogen producing bacteria, Clostridium butyricum, were immobilized in agar gel (2 per cent). The immobilized whole cells were employed for continuous production of hydrogen from alcohol factory waste waters. The hydrogen production rate became constant above BOD 1500 ppm when hydrogen production was performed with a batch system. The immobilized whole cells continuously produced hydrogen over a 20-day period. The amount of hydrogen produced was about 6 ml/min/kg wet gels. Hydrogen produced was supplied to the hydrogen-oxygen (air) fuel cells. The maximum cell voltage of cell I and II was about 0.55 and 0.66 V respectively when the flow rate of hydrogen was 6 ml/min. The limiting current density changed from 0.4 to 40 mA/cm2 as the resistance between the electrodes changed from 1 to 100 ohmz. The fuel cell was left on for 7 days and the current from 550 to 500 mA was obtained continuously over a 7 day period.

Ultramicrobiosensors for monitoring of neurotransmitters.

Carbon fiber electrodes are used to construct ultramicrobiosensors with 7-15 microns diameter. Electrochemical operations for preelectrolysis and measuring were examined for sensitive determination of hydrogen peroxide. Determination limit was 0.1 microM of hydrogen peroxide. Reproducible determination of hydrogen peroxide is possible even in samples containing albumin protein. A micro-acetylcholine sensor was fabricated by immobilizing acetylcholine esterase and choline oxidase on the carbon fiber by entrapment with PVA-SbQ. This sensor gave a linear calibration plot for the range from 0.1 to 1.0 mM with a linear correlation coefficient of 0.9842. A micro-glutamate sensor consisted of a platinized carbon fiber disk electrode modified with immobilized glutamate oxidase membrane. This sensor gave a linear calibration for the range 2 microM to 1.2 mM. Release of glutamate in the cerebellar cortex was detected after potassium stimulation.

Biosensors for environmental monitoring.

Environmental monitoring is of great importance for its protection. Conventional monitoring methods are often slow and complex and require expensive equipment, making them unsuitable for in situ, real-time monitoring of pollutants. Biosensors based on a combination of a biological sensing element and an electronic signal-transducing element are alternative methods to conventional ones. Biosensors have a number of advantages, such as high selectivity, high stability, and short response time. Various kinds of biosensors have been developed and employed for detection of pollutants such as phosphate, cyanide, and herbicides. Some of these have already been exploited as real-time monitoring in situ. In this article, some of the applications of biosensors for environmental control are described.

Electrochemical measurements of cell populations.

Determination of cell growth was carried out by a polarographic system. The system was constructed of two platinum electrodes, a saturated calomel electrode, and a thermistor electrode. Responses of the system to the dissolved oxygen, pH, and temperature were examined. Cell growth of S. cerevisiae and M. olivoasterospora was monitored continuously by this system. In addition, this polarographic system could be applied to the measurements of cell populations of the human cancer cell L-1210 and mouse leukocytes. The measureable range for these animal cells was approximately 10(3)--10(5) cells/ml.