Selective permeation of hydrogen peroxide through polyelectrolyte multilayer films and its use for amperometric biosensors.

A platinum electrode was coated with polyelectrolyte multilayer (PEM) films to prepare an amperometric hydrogen peroxide sensor which can be used in the presence of possible interferences such as ascorbic acid, uric acid, and acetaminophen. The PEM films were prepared on the surface of a Pt disk electrode by an alternate deposition of polycation and polyanion from the aqueous solutions through electrostatic force of attraction. The Pt electrodes coated with a poly(allylamine)/poly(vinyl sulfate) or poly(allylamine)/poly(styrenesulfonate) film were used successfully for detecting H2O2 selectively in the presence of the possible interfering agents. It was suggested that H2O2 can diffuse into the PEM film smoothly while the ascorbic acid, uric acid, and acetaminophen cannot penetrate the film by a size exclusion mechanism. On the other hand, the electrodes coated with PEM films containing poly(ethyleneimine) or poly(diallyldimethylammonium chloride) were not useful for the selective determination of H2O2. The results were rationalized based on the different permeability of the films due to the different molecular density or packing in the PEM films. The PEM film-coated electrode was useful for constructing glucose biosensors by coupling with glucose oxidase.

Glucose and lactate biosensors prepared by a layer-by-layer deposition of concanavalin A and mannose-labeled enzymes: electrochemical response in the presence of electron mediators.

An electrochemical response of glucose and lactate biosensors which were prepared by coating a platinum electrode with a thin film composed of concanavalin A and mannose-labeled glucose oxidase (GOx) or lactate oxidase (LOx) was evaluated in the presence of ferrocene derivatives as electron mediator. Both glucose and lactate biosensors showed catalytic current to glucose and lactate, respectively, in cyclic voltammetry, suggesting that the ferrocene derivatives can mediate electron transport smoothly from the reduced forms of GOx and LOx in the thin films to the electrode. Among the three kinds of ferrocene derivatives used, ferrocenylmethanol was found to be the most suitable electron mediator because of its low oxidation potential. The glucose and lactate sensors gave useful calibration graphs, in which higher detection limits were reached as compared with those observed when the sensors were operated in the absence of electron mediator.

Selective electrocatalytic oxidation of N-alkyl-N-methylanilines to N-alkylformanilides using nitroxyl radical.

Electrocatalytic oxidation of N-alkyl-N-methylanilines was studied using 4-benzoyloxy-2,2,6,6-tetramethyl-piperidinyl-N-oxyl as a nitroxyl radical. The reaction with N-alkyl-N-methylanilines led to direct formation of N-alkylformanilides in the presence of H2O in reaction media in adequate conversion (>75.8%), high current efficiency (>89.2%) and high selectivity (>93.8%).

Acceleration of fracture healing in nonhuman primates by fibroblast growth factor-2.

One of the greatest needs in the clinical bone field is a bioactive agent to stimulate bone formation. We previously reported that fibroblast growth factor-2 (FGF-2) exhibited strong anabolic actions on bone formation in models of rodents and dogs. Aiming at a clinical application, this study was undertaken to clarify the effect of a single local application of recombinant human FGF-2 on fracture healing in nonhuman primates. After a fracture was created at the midshaft of the right ulna of animals and stabilized with an intramedullary nail, gelatin hydrogel alone (n = 10) or gelatin hydrogel containing 200 microg FGF-2 (n = 10) was injected into the fracture site. Although 4 of 10 animals treated with the vehicle alone remained in a nonunion state even after 10 weeks, bone union was complete at 6 weeks in all 10 animals treated with FGF-2. Significant differences in bone mineral content and density at the fracture site between the vehicle and FGF-2 groups were seen at 6 weeks and thereafter. FGF-2 also increased the mechanical property of the fracture site. We conclude that FGF-2 accelerates fracture healing and prevents nonunion in primates, and therefore propose that it is a potent bone anabolic agent for clinical use.

Preparation of spatially ordered multilayer thin films of antibody and their binding properties.

Spatially ordered multilayer thin films containing anti-fluoresceinisothiocyanate (anti-FITC) were prepared on the surface of a quartz slide to study the binding properties of the multilayer films. A quartz slide was treated in solutions of avidin and biotin-labeled anti-FITC alternately and repeatedly to form multilayer thin films through a strong affinity between avidin and biotin. A spectrophotometric study revealed explicitly that the thin films thus prepared consisted of alternate monomolecular layers of avidin and biotin-labeled anti-FITC. The antibody retained its binding activity to antigen in the multilayer thin film, though the antigen could not access the antibody embedded deep in the multilayer film. Only the outermost four or five layers of antibody were involved in the binding of antigen.

Layer-by-layer construction of enzyme multilayers on an electrode for the preparation of glucose and lactate sensors: elimination of ascorbate interference by means of an ascorbate oxidase multilayer.

A layer-by-layer structure of enzyme multilayers composed of glucose oxidase (GOx) or lactate oxidase (LOx) and ascorbate oxidase (AOx) was prepared on the surface of a platinum electrode. The amperometric response to glucose or lactate was studied in the presence of ascorbic acid as a possible interference. An alternating and repeated deposition of avidin and the biotin-labeled enzymes resulted in the layer-by-layer structure of GOx/AOx and LOx/AOx multilayers. Optical and gravimetric measurements based on an ultraviolet-visible absorption spectroscopy and a quartz crystal microbalance revealed that the enzyme multilayers thus prepared consist of monomolecular layers of the proteins. The GOx/AOx and LOx/AOx enzyme multilayers were useful to eliminate ascorbic acid interference in the glucose and lactate biosensors, because ascorbic acid can be converted to an electrochemically inert form, dehydroascorbic acid, before being oxidized directly on the Pt electrode. Thus, the GOx/AOx or LOx/AOx multilayer-modified biosensors can be used to determine the normal blood level of glucose (5 mM) and lactate (1 mM) in the presence of a physiological level of ascorbic acid (0.1 mM). The effects of the number of the AOx layers and geometry of the enzyme layers in the multilayer on the performance characteristics of the biosensors are discussed.

Self-assembled biotinylated disulfide derivative monolayer on gold electrode for immobilizing enzymes.

Based on self-assembled biotinylated disulfide derivative monolayer on gold electrode, the sensors immobilized monolayer or multilayer membranes composed of avidin and biotinlabeled glucose oxidase (B.GOD) or of avidin-B.GOD complex (ABC) and B.COD were prepared. The present technique may be useful for controlling the enzyme content of the sensors in molecular level by repeating the deposition of enzyme layers. The sensors have the characteristics of shorter response time, higher sensitivity. The linear range is from 6.0 x 10(-6) - 5.0 x 10(-3) M. The sensor can be used for more than 1 month and can be reactivated. The sensor was used to determine glucose in human blood serum, and the results are satisfactory.

Monoclonal antibody #5-2-26 recognizes the phosphatase-sensitive epitope of rabies virus nucleoprotein.

We prepared monoclonal antibodies (MAbs) against the rabies virus N protein, among which one antibody (MAb 5-2-26) was shown to lack reactivity with the phosphatase-treated N protein. The MAb was able to recognize the sodium dodecyl sulfate (SDS)-denatured N protein. The MAb did not recognize the N-protein analogues produced in Escherichia coli (E. coli), indicating that the N-gene products were not normally processed in E. coli after translation. On the other hand, the MAb reacted normally with N-gene products produced in COS-7 cells, but not with those produced in the presence of K-252a (a protein kinase inhibitor of a broad spectrum). The MAb displayed weak cross-reactivity with the Triton-insoluble network structures composed of several components, while another phosphoprotein (M1) of the virus was not recognized at all. These results suggest that MAb 5-2-26 preferentially recognizes a phosphatase-sensitive linear epitope of N protein, which may enable further investigations to be conducted on the mechanism of N-protein phosphorylation and its role(s) in virus replication.

Effects of gender and gonadectomy on ACTH response to interleukin-1beta in the rat: comparison with the modulation of ACTH response to immobilization stress.

We examined the influence of gender and gonadectomy on the plasma adrenocorticotropin (ACTH) response to intravenous administration of human recombinant interleukin (IL)-1beta (3 microg/kg) in the rat. For comparison, we also examined whether gender and gonadectomy affect ACTH secretion after immobilization stress (a 30-min period), which is a nonimmunological stressor. IL-1beta induced a significantly higher ACTH response in females than in males, and this sexual difference was abolished by gonadectomy in both sexes. By contrast, ACTH secretion after immobilization was statistically the same in males and females, but tended to be higher in gonadectomized males than in gonadectomized females. These results may suggest a dissociative regulation by gonadal steroids of IL-1beta- and immobilization-induced ACTH responses in the rat. The sexual difference in ACTH response to IL-1beta may represent another example of the sexually dimorphic immunological activity, which is known to be higher in females than in males.

Adsorption behavior of serum albumin on electrode surfaces and the effects of electrode potential.

The adsorption behavior of serum albumin onto the surface of platinum, gold, and glassy carbon electrodes was studied in relation to the electrode potential, by using cyclic voltammetry and a quartz-crystal microbalance. The kinetics of adsorption was significantly dependent on the electrode potential. The adsorption was highly accelerated by the application of positive potential to the electrode, suggesting an electrostatic interaction between the negatively charged albumin molecules and the positively polarized electrode as the origin of the accelerated adsorption. The adsorption of albumin on the electrodes was irreversible with respect to dilution of the albumin solution, while the quartz-crystal microbalance data showed that albumin forms a monomolecular layer on the electrode surface. Protein adsorption on electrode surface in serum was also examined.

Modification of a glassy carbon electrode with diols for the suppression of electrode fouling in biological fluids.

The surface of a glassy carbon (GC) electrode was modified covalently with ethyleneglycol, diethyleneglycol, 1,2-propanediol, and 1,3-propanediol by electrochemical oxidation in order to suppress the electrode fouling originating from non-specific adsorption of serum proteins. Human serum albumin (HSA) was adsorbed significantly on the surface of a bare GC electrode, which was monitored by cyclic voltammetry in the presence of Fe(CN)6(4)-/Fe(CN)6(3)-ions. In contrast, the diol-modified GC electrodes were scarcely fouled in HSA solution and even in human serum. The results were explained reasonably based on the hydrophilic nature of the diol-modified GC surface.

Controlled deposition of glucose oxidase on platinum electrode based on an avidin/biotin system for the regulation of output current of glucose sensors.

A facile method for the regulation of enzyme loading on an electrode surface has been studied using avidin and biotinylated glucose oxidase (GOx). It was demonstrated that an alternate and repeated deposition of avidin and biotinylated GOx gives a protein thin film probably composed of avidin monolayers and biotinylated GOx monolayers which are connected with each other through strong affinity between avidin and biotin moieties of the enzyme (binding constant, 10(15) M-1). Amperometric response of the glucose sensors constructed by this method was controlled stepwise and rather precisely by regulating the number of GOx layers deposited (or the loading of GOx). For example, the output current of the sensors to 1 mM glucose was enhanced to ca. 1300 and 2800 nA after deposition of 10 and 20 layers of GOx, respectively, as compared with 110 nA for the monolayer GOx sensor. The enhanced response contributed to the extension of the dynamic range of the sensors, especially at a lower glucose concentration. The response time of the sensors was satisfactorily fast (ca. 20 s), irrespective of the number of GOx layers.

Electrochemically accelerated adsorption of serum albumin on the surface of platinum and gold electrodes.

Adsorption of serum albumin on the surface of platinum and gold electrodes was highly accelerated by the application of a constant potential to the electrodes. The accelerated adsorption was significant at the electrode potential of 0.5-1.0 V vs. Ag/AgCl, even in the diluted solution of albumin (0.01%).

Neuropeptide Y potentiates the luteinizing hormone (LH) response to LH-releasing hormone in men.

It has been demonstrated in several animal species that neuropeptide Y (NPY) exerts a modulatory effect on luteinizing hormone (LH) secretion. However, whether NPY plays a similar role also in humans has yet to be determined. Therefore, in this study we examined the effect of human NPY on the anterior pituitary hormone secretion in 6 normal men. Intravenous bolus injection of 100 micrograms of human NPY alone did not affect the secretion of any anterior pituitary hormone or cortisol. However, when NPY (100 micrograms) was administered simultaneously with LH-releasing hormone (LHRH, 100 micrograms), a significant potentiation was observed for LHRH-induced LH secretion. Similarly, follicle-stimulating hormone (FSH) response to LHRH was slightly potentiated by the coadministration of NPY, although this effect was not statistically significant. This is the first study to demonstrate that NPY can augment the LHRH-induced LH secretion in humans.

Photo-switchable ion and enzyme sensors. Photoinduced potentiometric response of glassy carbon electrode coated with polymer or polymer/enzyme dual membrane.

Photo-switchable ion and enzyme sensors were fabricated by the use of glassy carbon electrode coated with nonactindoped or enzyme modified poly(vinyl chloride) (PVC) membranes. The ion sensor with nonactin-doped PVC membrane, which contained spirobenzopyran as the photosensitive dye, exhibited a potentiometric photoresponse to NH4+ ion in the solution. The dynamic range of the NH4+ ion sensor was 10(-7)--10(-3) M. Urea, adenosine, and asparagine sensors were prepared by coating the surface of the NH4+-ion sensor with urease, adenosine deaminase, and asparaginase membranes, respectively. These enzyme sensors could be used for determining the substrates at the micro mole level. The performance characteristics of these sensors were compared with those previously prepared membrane electrode sensors.

Enzyme sensors based on an ion-sensitive field effect transistor coated with Langmuir-Blodgett membranes. Use of polyethyleneimine as a spacer for immobilizing alpha-chymotrypsin.

A highly branched polyethyleneimine (PEI) was used as a spacer for immobilizing alpha-chymotrypsin on the surface of Langmuir-Blodgett (LB) membranes which were deposited on the gate of an ion-sensitive field effect transistor (ISFET). alpha-Chymotrypsin could be covalently immobilized through the glutaraldehyde-activated PEI on the LB membrane-coated ISFET. The alpha-chymotrypsin-modified ISFET showed a potentiometric response to the substrate at concentrations of more than 0.1 mM. Some performance characteristics of the sensor, such as pH response, response time, and long-term stability were examined.