Tomonori Hoshi

Amperometric uric acid sensors based on polyelectrolyte multilayer films.

Uricase (UOx) and polyelectrolyte were used for preparation of a permselective multilayer film and enzyme multilayer films on a platinum (Pt) electrode, allowing the detection of uric acid amperometrically. The polyelectrolyte multilayer (PEM) film composed of poly(allylamine) (PAA) and poly(vinyl sulfate) (PVS) were prepared via layer-by-layer assembly on the electrode, functioning as H(2)O(2)-selective film. After deposition of the permselective film (PAA/PVS)(2)PAA, UOx and PAA were deposited via layer-by-layer sequential deposition up to 10 UOx layers to prepare amperometric sensors for uric acid. Current response to uric acid was recorded at +0.6 V vs. Ag/AgCl to detect H(2)O(2) produced from the enzyme reaction. The response current increased with increasing the number of UOx layers. Even in the presence of ascorbic acid, uric acid can be detected over the concentration range 10(-6)-10(-3) M. The response current and deposited amount of UOx were affected by deposition bath pH and the addition of salt. The deposition of PAA/UOx film prepared in 2 mg ml(-1) solution (pH 11) of PAA with NaCl (8 mg ml(-1)) and 0.1 mg ml(-1) solution (pH 8.5) of UOx with borate (100 mM) resulted in an electrode which shows the largest response to uric acid. The response of the sensor to uric acid was decreased by 40% from the original activity after 30 days.

Avidin-Biotin System-Based Enzyme Multilayer Membranes for Biosensor Applications: Optimization of Loading of Choline Esterase and Choline Oxidase in the Bienzyme Membrane for Acetylcholine Biosensors

The loading of choline esterase (ChE) and choline oxidase (ChOx) in the enzyme membrane of an acetylcholine biosensor was optimized based on a layer-by-layer construction of the bienzyme layers on the surface of a platinum (Pt) and a Pt-black electrode. To this goal, ChE and ChOx were tagged with biotin residues, so that the enzymes could be built into the multilayer assemblies composed of monomolecular layers of the enzymes and avidin. The ChE/ChOx bienzyme multilayer-modified electrodes showed amperometric response to acetylcholine in solution, showing that ChE and ChOx catalyzed hydrolysis reaction of acetylcholine and an oxidation reaction of resulting choline, respectively, successively in the bienzyme layer. It was found that the acetylcholine sensor which is modified with 10-layer ChOx exhibited its maximum response to acetylcholine when additional 2 layers of ChE were added to the surface of the ChOx layer. This optimum ratio of ChE to ChOx in the bienzyme layer was reasonably explained in terms of the relative values of catalytic activity of the enzymes. The usefulness of the layer-by-layer deposition technique in optimizing the enzyme loading in bienzyme system is discussed in detail.

Enzyme sensors prepared by layer-by-layer deposition of enzymes on a platinum electrode through avidin–biotin interaction

Abstract An alternate and repeated deposition of avidin and biotin-labeled glucose oxidase (GOx) or lactate oxidase (LOx) on a solid surface gave protein multilayers through a strong binding between avidin and biotin. Spectrophotometric and gravimetric techniques using UV absorption and a quartz-crystal microbalance revealed that the protein multilayers are composed of avidin monolayers and enzyme monolayers which in turn are built in a layer-by-layer structure. The GOx and LOx enzymes were found to be catalytically active in the multilayer films, by means of a cyclic voltammetry. The response characteristics of the GOx- and LOx-modified sensors are discussed.

Construction of positively-charged layered assemblies assisted by cyclodextrin complexation

A beta-cyclodextrin dimer is found to be effective in preparing a layer-by-layer architecture of positively charged ferrocene-appended poly(allylamine) presumably on the basis of strong beta-cyclodextrin-ferrocene host-guest interaction.

Avidin—biotin complexation for enzyme sensor applications

Abstract The use of an avidin—biotin system in the preparation of enzyme sensors is described. The biotin-labelled enzymes can be immobilized on the avidin-modified electrode surface through avidin—biotin complexation. The various techniques for the surface derivatization with biotin and avidin and for the coupling with enzymes are cited. The possibility of constructing a protein architecture on the electrode is also discussed: this is based on the non-covalent interaction of avidin and biotin.

Electrochemical deposition of avidin on the surface of a platinum electrode for enzyme sensor applications

Avidin was electrochemically deposited on a platinum electrode without loss of binding activity to biotin, and biotin-labeled glucose oxidase was immobilized on the electrode through avidin-biotin binding. The electrodes thus prepared displayed excellent properties as a glucose sensor. The oxidation current is proportional to glucose concentration from 10−4 to 10−2 M, and the sensor could be used for more than 3 months.

Use of Con A and mannose-labeled enzymes for the preparation of enzyme films for biosensors

Abstract Concanavalin A (Con A) and mannose-labeled enzymes are used to prepare Con A/enzyme composite thin films, in which enzymes are catalytically active. Preparation and characterization of the thin films and their use for glucose and lactate biosensors are discussed.

Preparation of enzyme multilayers on electrode surface by use of avidin and biotin-labeled enzyme for biosensor applications

Abstract Multilayer membranes composed of avidin and biotin-labeled glucose oxidase (B-GOD) or of an avidin—B-GOD complex and B-GOD were prepared on an indium tin oxide (ITO) electrode, based on the high affinity between avidin and B-GOD. The B-GOD-modified ITO electrodes can be used to determine glucose electrochemically. The magnitude of the output signal (i.e., electric current) of the electrodes depended clearly on the number of enzyme layers.

Multilayer membranes via layer-by-layer deposition of ascorbate oxidase and Au nanoparticles on the Pt electrode for reduction of oxidation current derived from ascorbate

A glass plate was alternately immersed in an Au colloid (10nm phi) and an ascorbate oxidase (AO(x)) solution (0.1mg/mL, pH 6.8). Absorbance at 530nm originating from the Au particles increased with the increasing number of depositions. The AO(x) activity of the plate also increased as the plate was immersed in the AO(x) solution. These results suggested that a multilayer membrane via the layer-by-layer deposition of AO(x) and Au nanoparticles was formed on the glass plate. AO(x) was also deposited on a Pt disk electrode using the same process. Using the (AO(x)/Au)(10) modified electrode, the oxidation current of ascorbic acid (0.1mM) decreased to 19% versus the unmodified electrode.

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.