Yukari Sato

Glucose oxidase/polyion complex-bilayer membrane for elimination of electroactive interferents in amperometric glucose sensor

Abstract An amperometric glucose-sensing electrode was prepared by immobilizing glucose oxidase (GOx) on a polyion complex membrane. First, a monolayer of 3-mercaptopropionic acid (MPA) was made on the surface of a gold electrode by immersing it in an ethanol solution containing MPA. Aqueous solutions of poly- l -lysine and poly-4-styrenesulfonate were successively placed on the electrode surface and allowed to dry. A GOx layer was then formed on the poly- l -lysine/poly-4-styrenesulfonate-complex layer by crosslinking the enzyme by the addition of a glutaraldehyde solution. The polyion complex layer was effective for eliminating electrochemical interferents such as l -ascorbic acid, uric acid and acetaminophen, whereas the hydrogen peroxide produced through the GOx-catalyzed reaction permeated rapidly through the layer. This resulted in a rapid response (100% response in l -ascorbic acid to that for the same concentration of glucose was 0.07). The electrode was applied to the assay of glucose in beverages and sera, and could be used for more than two months.

Electrochemical properties of the 2-mercaptohydroquinone monolayer on a gold electrode. Effect of solution pH, adsorption time and concentration of the modifying solution

The redox properties of an adsorbed mercaptohydroquinone (H 2 QSH) monolayer on a gold electrode in both buffered and unbuffered solutions over a very wide pH range (0-12.7) are investigated in detail and are compared with those of free quinone (Q)-hydroquinone (H 2 Q) species in solution at an unmodified gold electrode. Reversible waves due to the redox process of the immobilized Q-H 2 Q groups are observed. The redox potentials vary with pH with a slope of -60 mV pH -1 , showing that the reaction proceeds as a 2-electron-2-proton reaction. The redox potentials of the surface-attached H 2 QSH are more negative than those of free H 2 Q in solution. The redox behavior becomes complex in neutral unbuffered solution and two redox waves are observed. Several possibilities are considered for the origins of these redox waves based on the results of detailed experiments including sweep rate dependence and the effect of proton concentration. The effect of the adsorption time and the concentration of H 2 QSH in the modifying solution on the redox potential of the adsorbed H 2 QSH monolayer on a gold electrode are also studied. The number of adsorbed molecules increases as the concentration of H 2 QSH becomes higher and the dipping time becomes longer. The redox potential of the adsorbed H 2 QSH is affected by both the adsorption time and the concentration of H 2 QSH in the modifying solution and becomes more negative as the coverage of H 2 QSH increases.

In situ STM imaging of individual molecules in two-component self-assembled monolayers of 3-mercaptopropionic acid and 1-decanethiol on Au(111)

Two-component self-assembled monolayers composed of 3-mercaptopropionic acid (MPA) and 1-decanethiol (CH3(HC2)9SH:C10SH) on Au(111) were investigated with in situ scanning tunneling microscopy (STM) and cyclic voltammetry, where the monolayers I and II were prepared in 0.1 mM ethanol+water solutions with the ratios of MPA:C10SH=95:5 and 90:10, respectively. In situ STM images revealed that both monolayer I and II consisted of phase-separated domains with molecularly ordered structures, each of which was predominantly formed by one of the constituent molecules. Based on the STM images, the surface fractions of constituent molecules were evaluated as MPA:C10SH=0.61:0.28 for monolayer I, and MPA:C10SH=0.34:0.57 for monolayer II. Cyclic voltammograms of the reductive desorption of the thiols provided the surface fractions of MPA:C10SH=0.69:0.31 for monolayer I, and MPA:C10SH=0.41 and 0.59 for monolayer II, which were in good agreement with the results obtained from STM images. Molecular resolution imaging allowed us to visualize the individual MPA and C10SH molecules and to determine the interfacial structures in the molecularly ordered domains of the phase-separated monolayer. The C10SH domains in the monolayer exhibited ordered phases with densely packed (√3×√3)R30° and p(3×2√3R−30°) structures, which are well-characterized structures for alkanethiol monolayers. On the other hand, a completely different molecular arrangement of MPA defined as a (3×3) structure was consistently observed in the MPA domains, where the molecular arrangement is almost the same as that of (√3×√3)R30° but intermolecular hydrogen bonding is thought to exist in the three neighboring MPA molecules located around the corner of the (3×3) unit cell. It was demonstrated that individual molecules of the monolayer constituents were successfully imaged in the two-component, phase-separated monolayer in solution.

Amperometric determination of pyruvate, phosphate and urea using enzyme electrodes based on pyruvate oxidase-containing poly(vinyl alcohol)/polyion complex-bilayer membrane

Abstract An amperometric pyruvate-sensing electrode was prepared by immobilizing pyruvate oxidase (PyOx) on a polyion complex membrane. First, aqueous solutions of poly- l -lysine and poly(4-styrenesulfonate) were successively placed on a mercaptopropionic acid-modified gold surface and allowed to dry. A photo-crosslinked poly(vinyl alcohol) layer containing PyOx was then formed on the poly- l -lysine/poly(4-styrenesulfonate)-complex layer. The polyion complex layer was effective for eliminating electrochemical interferents such as l -ascorbic acid, uric acid, l -cysteine and acetaminophen, whereas the hydrogen peroxide produced through the PyOx-catalyzed reaction permeated easily through the layer. This resulted in a high sensitivity (detection limit, 50 nM) and a low interference level (e.g. the ratio of response for l -ascorbic acid to that for the same concentration of pyruvic acid, 0.18). The electrode could be used for determining phosphoric acid (detection limit, 0.2 μM), since PyOx consumes phospholic acid as the co-substrate during the course of pyruvate oxidation. Further, an amperometric urea-sensing electrode (detection limit, 0.5 μM) was prepared by coupling the phosphate-sensing system with urea amidolyase which catalyzes an ATP-consuming urea hydrolyzation.

RAPID MEASUREMENT OF TRANSAMINASE ACTIVITIES USING AN AMPEROMETRIC L-GLUTAMATE-SENSING ELECTRODE BASED ON A GLUTAMATE OXIDASE-POLYION COMPLEX-BILAYER MEMBRANE

Abstract An amperometric l -glutamate-sensing electrode was prepared by immobilizing glutamate oxidase (GlOx) on a polyion complex layer-modified electrode. First, a monolayer of 3-mercaptopropionic acid was made on the surface of a gold electrode by immersing it in an ethanol solution containing the modifier. Next, aqueous solutions of poly- l - lysine and poly(4-styrenesulfonate) were successively placed on the electrode surface and allowed to dry. Finally, a GlOx layer was formed on the poly- l- lysine/poly(4-styrenesulfonate)-complex layer by crosslinking the enzyme by the addition of a glutaraldehyde solution. The use of thin bilayer system with the inner, polyion complex membrane, which showed permselectivity based on the solute size with the molecular cut-off of ≈100, brought high performance characteristics to the l- glutamate-sensing electrode; it showed high sensitivity (detection limit, 20 nM), rapid response (100% response time, 3 s), low interferential level (the ratio of response for l -ascorbic acid to that for the same concentration of l -glutamic acid, 8×10−2), and high stability (usable for more than a month). The bilayer-based electrode was useful for the rapid measurement of glutamate–oxaloacetate transaminase (GOT) and glutamate–pyruvate transaminase (GPT) in serum sample: each transaminase (0.2–1000 U l−1) could be determined within 10 s.

High-throughput flow-injection analysis of glucose and glutamate in food and biological samples by using enzyme/polyion complex-bilayer membrane-based electrodes as the detectors

The concentration of glucose was determined by a combination of flow injection analysis (FIA) with amperometric enzyme sensor detection. The enzyme sensor was prepared by immobilizing glucose oxidase on an electrode coated with a polyion complex layer consisting of poly-L-lysine and poly(4-styrenesulfonate). The inner, polyion complex layer was useful for preventing electrochemical interferents (e.g., L-ascorbic acid, uric acid and acetaminophen) from reaching the electrode surface, which was effective for reducing the interferential responses upon the injections of biological and food samples. The sensor-based system could be used for the determination of glucose from 10 microM to 3 mM with the sampling rate of 180 h-1, and was stable for more than 2 months. An FIA system for determining L-glutamic acid (3 microM-0.5 mM) was also prepared by using an enzyme electrode based on a glutamate oxidase/polyion complex-bilayer as the detector.

Coverage dependent behavior of redox reaction induced structure change and mass transport at an 11-ferrocenyl-1-undecanethiol self-assembled monolayer on a gold electrode studied by an in situ IRRAS–EQCM combined system

The potential dependent structure change and mass transport at an 11-ferrocenyl-1-undecanethiol (FcC11SH) self-assembled monolayer on a gold electrode with various coverages were investigated by simultaneous Fourier transform infrared reflection absorption spectroscopy (IRRAS) and electrochemical quartz crystal microbalance (EQCM) measurements in 0.1 M HClO4 solution. As soon as the terminal ferrocene group was oxidized to a ferricenium cation (Fc+), a number of upward and downward IRRAS bands were observed and the surface mass was increased. The intensity of the IRRAS bands corresponded well with the mass change. This behavior is attributed to the redox reaction induced orientation change in the FcC11SH monolayer and ion pair formation between the Fc+ cation and the perchlorate anion in solution. The FcC11SH monolayer with a lower coverage was less stable and was decomposed at a less positive potential than that with a high coverage. Models are proposed to explain the coverage dependent behavior of the FcC11SH monolayer induced by the redox reaction of the terminal ferrocene moiety.

Electrochemical responses of cytochrome c on a gold electrode modified with mixed monolayers of 3-mercaptopropionic acid and n-alkanethiol☆

The electrochemical behavior of cytochrome c on gold electrodes modified in solution containing a mixture of 3-mercaptopropionic acid and n-alkanethiol having various chain lengths was investigated and compared with that of another mixed monolayer system, bis(4-pyridyl) disulfide and n-alkanethiol, previously reported. The peak current decreased with the increase of the ratio of n-alkanethiol in the 3-mercaptopropionic acid + n-alkanethiol mixture, as was observed in the case of bis(4-pyridyl) disulfide + n-alkanethiol mixture. The n-alkanethiol with a longer chain was more effective in blocking the electron exchange between cytochrome c and the electrode than that with a shorter chain, in contrast to the results observed at the mixed monolayer of bis(4-pyridyl) disulfide and n-alkanethiol. This observation reflects the fact that adsorbed molecules were well mixed with each other in the case of the 3-mercaptopropionic acid + n-alkanethiol mixed monolayers, while patched domains of pyridyl thiolate were formed in the case of the mixed monolayers of bis(4-pyridyl) disulfide and n-octadecyl mercaptan. The electrochemical behavior of the gold electrodes modified with the mixed monolayer of 3-mercaptopropionic acid and n-alkanethiol in the solution containing Fe(CN)64−3− supports the above observation. Reductive desorption of the mixed monolayer was carried out in alkaline solution to determine the composition and to evaluate the degree of mixing of the two components.

Surface modification of GC and HOPG with diazonium, amine, azide, and olefin derivatives.

Surface modification of glassy carbon (GC) and highly oriented pyrolytic graphite (HOPG) was carried out with diazonium, amine, azide, and olefin derivatives bearing ferrocene as an electroactive moiety. Features of the modified surfaces were evaluated by surface concentrations of immobilized molecule, blocking effect of the modified surface against redox reaction, and surface observation using cyclic voltammetry and electrochemical scanning tunneling microscope (EC-STM). The measurement of surface concentrations of immobilized molecule revealed the following three aspects: (i) Diazonium and olefin derivatives could modify substrates with the dense-monolayer concentration. (ii) The surface concentration of immobilized amine derivative did not reach to the dense-monolayer concentration reflecting their low reactivity. (iii) The surface modification with the dense-monolayer concentration was also possible with azide derivative, but the modified surface contained some oligomers produced by the photoreaction of azides. Besides, the blocking effect against redox reaction was observed for GC modified with diazonium derivative and for HOPG modified with diazonium and azide derivatives, suggesting fabrication of a densely modified surface. Finally, the surface observation for HOPG modified with diazonium derivative by EC-STM showed a typical monolayer structure, in which the ferrocene moieties were packed densely at random. On the basis of those results, it was demonstrated that surface modification of carbon substrates with diazonium could afford a dense monolayer similar to the self-assembled monolayer (SAM) formation.

Formation and characterization of aromatic selenol and thiol monolayers on gold: in-situ IR studies and electrochemical measurements

Adsorption processes and electrochemical properties of benzeneselenol and benzenethiol on gold electrodes were investigated by surface-enhanced IR absorption spectroscopy (SEIRAS) and electrochemical measurements. From the IR spectroscopic measurements, it was confirmed that benzeneselenol molecules adsorbed on the gold surface as benzeneselenolate–Au. SEIRAS measurements revealed that benzeneselenol took a much longer time to adsorb on a gold surface than benzenethiol. Electrochemical reductive desorption of these monolayers in alkaline solution proved that benzeneselenoate chemisorbed on gold surfaces were more stable compared to benzenethiolate, since the reduction peak potential of benzeneselenolate shifted to more than 200 mV negative potential than that of benzenethiolate. The total amounts of adsorbed benzeneselenol molecules were smaller than the case of benzenethiol.