Jun-ichi Anzai

pH- and sugar-sensitive layer-by-layer films and microcapsules for drug delivery ☆

The present review provides an overview on the recent progress in the development of pH- and sugar-sensitive layer-by-layer (LbL) thin films and microcapsules in relation to their potential applications in drug delivery. pH-sensitive LbL films and microcapsules have been studied for the development of peptide and protein drug delivery systems to the gastrointestinal tract, anti-cancer drugs to tumor cells, anti-inflammatory drugs to inflamed tissues, and the intracellular delivery of DNA, where pH is shifted from neutral to acidic. pH-induced decomposition or permeability changes of LbL films and microcapsules form the basis for the pH-sensitive release of drugs. Sugar-sensitive LbL films and microcapsules have been studied mainly for the development of an artificial pancreas that can release insulin in response to the presence of glucose. Therefore, glucose oxidase, lectin, and phenylboronic acid have been used for the construction of glucose-sensitive LbL films and microcapsules. LbL film-coated islet cells are also candidates for an artificial pancreas. An artificial pancreas would make a significant contribution to improving the quality of life of diabetic patients by replacing repeated subcutaneous insulin injections.

Layer-by-layer construction of protein architectures through avidin–biotin and lectin–sugar interactions for biosensor applications

AbstractIn this review, the preparation and properties of protein architectures constructed by layer-by-layer (LbL) deposition through avidin–biotin and concanavalin A (Con A)–sugar interactions are discussed in relation to their use for optical and electrochemical biosensors. LbL films can be constructed through the alternate deposition of avidin and biotin-labeled enzymes on the surfaces of optical probes and electrodes. The enzymes retain their catalytic activity, resulting in the formation of optical and electrochemical biosensors. Alternatively, Con A can be used to construct enzyme-containing LbL films and microcapsules using sugar-labeled enzymes. Some enzymes such as glucose oxidase and horseradish peroxidase can be used for this purpose without labeling with sugar, because these enzymes contain intrinsic hydrocarbon chains on their molecular surfaces. The Con A/enzyme LbL architectures were successfully used to develop biosensors sensitive to specific substrates of the enzyme. In addition, Con A-based films can be used for the optical and electrochemical detection of sugars. FigureSugar-induced decomposition of Con A/glycogen LbL film for the electrochemical sugar sensing

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.

Amperometric choline biosensors prepared by layer-by-layer deposition of choline oxidase on the Prussian blue-modified platinum electrode

An amperometric choline biosensor was developed by immobilizing choline oxidase (ChOx) in a layer-by-layer (LBL) multilayer film on a platinum (Pt) electrode modified with Prussian blue (PB). 6-O-Ethoxytrimethylammoniochitosan chloride (EACC) was used to prepare the ChOx LBL films. The choline biosensor was used at 0.0V versus Ag/AgCl to detect choline and exhibited good characteristics such as relative low detection limit (5x10(-7)M), short response time (within 10s), high sensitivity (88.6muAmM(-1)cm(-2)) and a good selectivity. The results were explained based on the ultrathin nature of the LBL films and the low operating potential that could be due to the efficient catalytic reduction of H(2)O(2) by PB. In addition, the effects of pH, temperature and applied potential on the amperometric response of choline biosensor were evaluated. The apparent Michaelis-Menten constant was found to be (0.083+/-0.001)x10(-3)M. The biosensor showed excellent long-term storage stability, which originates from a strong adsorption of ChOx in the EACC multilayer film. When the present choline biosensor was applied to the analysis of phosphatidylcholine in serum samples, the measurement values agreed satisfactorily with those by a hospital method.

Self-assembled film of hydrophobins on gold surfaces and its application to electrochemical biosensing

Hydrophobins are small fungal proteins which self-assemble on interfaces and significantly change the surface wettability. The self-assembled film of hydrophobin HFBI on a gold surface improved the surface hydrophilicity with water contact angle changing from 73.8+/-1.8 degrees to 45.3+/-1.4 degrees . A quartz crystal microbalance (QCM) analysis indicated that the HFBI coverage density on a gold surface was 588 ng cm(-2), and the self-assembled film remained stable under different pH values ranging from 1 to 13. A hydrophilic protein such as choline oxidase (ChOx) was then successfully immobilized on the HFBI modified gold surface. To evaluate the bioactivity of immobilized enzyme, an amperometric choline biosensor was constructed based on the Gold/HFBI/ChOx electrode, which produced as large as 4578.27 nA response current by 0.238 microg immobilized ChOx, when saturated by choline substrate. Comparing with our choline biosensors previously reported, the HFBI self-assembled film exhibited excellent capability to preserve the bioactivity of ChOx, hence a great potential in electrochemical biosensing is suggested.

Enantioselective electrocatalytic oxidation of racemic sec-alcohols using a chiral 1-azaspiro[5.5]undecane-N-oxyl radical

Nitroxyl radical (6S,7R,10R)-4-acetylamino-2,2,7-trimethyl-10-isopropyl-1-azaspiro[5.5]-undecane-N-oxyl reveals a reversible redox peak in cyclic voltammetry at + 0.62 V vs. Ag/AgCl. A preparative electrocatalytic oxidation of racemic sec-alcohols on the nitroxyl radical yielded mixtures of 51.4 – 63.9 % ketones and 36.1 – 48.6 % alcohols by 10 h of electrolysis. The current efficiency and turnover number of the reactions were 85.6 – 87.9 % and 20.6 – 25.6, respectively. The enantiopurity of the remaining (R)-isomers was 50 – 70 % and the S values as a selective factor was 4.1 – 4.6.

An Unmediated Hydrogen Peroxide Sensor Based on a Hemoglobin-sds Film Modified Electrode

ABSTRACT An unmediated hydrogen peroxide sensor is designed in this paper by employing a hemoglobin-SDS film modified electrode. Hemoglobin exhibits direct (unmediated) electrochemistry at the modified electrode. The protein also shows elegant catalytic activity towards the electrochemical reduction of hydrogen peroxide. Consequently, a prototype hydrogen peroxide sensor is prepared. Under optimum conditions, this sensor provides a linear response over the hydrogen peroxide concentrations in the range of 1×10-5∼1×10-4 mol/L. The detection limit was 2×10-6 mol/L The relative standard deviation was 4.2% for 6 successive determinations of the hydrogen peroxide at 1×10-5 mol/L. This configuration is shown to be sensitive, stable and easily fabricated. It might be useful in the biological and industrial fields.

Quartz-crystal microbalance and cyclic voltammetric studies of the adsorption behaviour of serum albumin on self-assembled thiol monolayers possessing different hydrophobicity and polarity

Abstract The adsorption behaviour of bovine serum albumin (BSA) on the surface of four different types of thiol monolayer was studied using a quartz-crystal microbalance (QCM) and cyclic voltammetry (CV). BSA was bound to the thiol monolayers with different efficiencies, depending on their surface properties. BSA was adsorbed more efficiently to hydrophobic thiol monolayers than to hydrophilic thiol monolayers. The electrostatic force of attraction or repulsion between the BSA molecules and the monolayer surface did not play a primary role in determining the adsorption behaviour of BSA. The binding of BSA was highly accelerated around its isoelectric point (pH 5.0).

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.