Self-gelatinizable graft copolymer of poly(vinyl alcohol) with 4-vinylpyridine as an immobilization matrix for the construction of a tyrosinase-based amperometric biosensor

A tyrosinase-based amperometric biosensor using a self-gelatinizable graft copolymer of poly(vinyl alcohol) with 4-vinylpyridine (PVA-g-PVP) as an immobilization matrix was constructed. The 4-vinylpyridine component of PVA-g-PVP enhances the adherence to a glassy carbon electrode surface. The content of 4-vinylpyridine in this immobilization matrix plays a key role in retaining the activity of tyrosinase. A simple, milder method was adopted by simply syringing the copolymer-tyrosinase aqueous solution on to the electrode surface and allowing water to evaporate at 4 degrees C in a refrigerator. Several parameters, including copolymer composition; pH, applied potential and enzyme membrane composition, ware optimized. The enzyme membrane composition can be varied to obtain higher sensitivity or a wider linear detection range. The biosensor was used for the determination of phenol, p-cresol and catechol. The biosensor exhibited excellent reproducibility, stability and sensitive response and can be used in flow injection analysis. The biosensor showed an extended linear range in hydrophilic organic solvents and it can be used in monitoring organic reaction processes. The analytical performance demonstrated this immobilization matrix is suitable for the immobilization of tyrosinase.

Functionalized inorganic-organic composite material derivated by sol-gel for construction of mediated amperometric hydrogen peroxide biosensor

A novel functionalized inorganic-organic hybrid material with cation exchange property was prepared by sol-gel method. The H2O2 biosensor was fabricated by simply dipping the horseradish peroxidase-containing functionalized membrane modified electrode into Meldola's blue (MDB) solution. MDB was adsorbed and firmly immobilized within the membrane. The electrochemical behavior of MDB incorporated in the membrane was more reversible compared with that of the solution species and suitable as mediator for the horseradish peroxidase. The response time was less than 25 s. Linear range is up to 0.6 mM (COH. coeff. 0.9998) with detection Limit of 9 x 10(-7) M. High sensitivity of 75 nA mu M cm(-2) was obtained due to high MDB-loading. The biosensor exhibited a good stability. (C) 1999 Elsevier Science B.V. All rights reserved.

Self-gelatinizable copolymer immobilized glucose biosensor based on Prussian Blue modified graphite electrode

A novel poly(vinyl alcohol) grafting 4-vinylpyridine self-gelatinizable copolymer was adapted to immobilize glucose oxidase. The reduction of hydrogen peroxide (H2O2) was detected at a Prussian Blue (PB) modified graphite electrode. A stable and sensitive glucose amperometric biosensor is described. The copolymer is a good biocompatible polymer in which the glucose oxidase retains high activity. Moreover, the copolymer can adhere firmly to the inorganic PB membrane. The sensor showed an apparent Michaelis-Menten constant of 18 +/- 0.2 mM and a maximum current density of 1.14 mu A cm(-2) mM(-1). The linear range is from 5 mu M to 4.5 mM glucose and the detection limit is 0.5. mu M glucose. The catalytic efficiency of PB for the reduction of H2O2 is higher than that for the oxidation of H2O2. Glucose concentrations in serum samples from healthy persons and diabetic patients were determined using the sensor. The results compared well with those provided by the hospital using a spectroscopy method.

Amperometric glucose biosensor based on sol-gel organic-inorganic hybrid material

A new type of sol-gel organic-inorganic hybrid material was developed and used for the production of biosensors. This material is composed of silica sol and a grafting copolymer of poly(vinyl alcohol) with 4-vinylpyridine. It prevents the cracking of conventional sol-gel-derived glasses and eliminates the swelling of the hydrogel. The optimum composition of the hybrid material was first examined, and then glucose oxidase was immobilized in this matrix to demonstrate its application. The characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. The biosensor exhibited a series of good properties: high sensitivity (600 nA mmol(-1)L(-1)), short response time (11 s) and remarkable long-term stability in storage (at least 5 months). In addition, the characteristics of the second-generation biosensor with the use of tetrathiafulvalene as a mediator mere discussed.

Amperometric biosensor for tyrosinase inhibitors in a pure organic phase

The determination of benzoic acid, thiourea and 2-mercaptoethanol in three pure organic solvents, viz., chloroform, chlorobenzene and 1,2-dichlorobenzene, by using an amperometric cryohydrogel tyrosinase biosensor is described. Measurements were carried out with phenol as the enzyme substrate. Kinetic parameters (K-i and I-50) were determined in the three solvents for various inhibitors. The sensor showed the most sensitive measurements to these inhibitors in pure chloroform. The solvent-induced deviation of the biosensor to thiourea was evaluated by means of Hill coefficients. The smallest deviation as observed in 1,2-dichlorobenzene, owing to the high hydrophobicity of this solvent. The nature of the inhibition process and its reversibility mere also examined.

Cryoimmobilized enzyme for biosensor construction

A new immobilization material and an immobilization method for a glucose sensor with HEFc (hydroxyethylferrocene) as mediator is described. In the course of three months, the enzyme electrode shows almost no deterioration in its response characteristics. The response time is less than 30 s. The electrode has a wide linear range up to 10 mmol l(-1) with good repeatability. The kinetic parameters have also been calculated.

Cyro-hydrogel for the construction of a tyrosinase-based biosensor

A new immobilization method for construction of a tyrosinase based biosensor is described. A simple physical freezing technique was adopted for preparation. The immobilized enzyme yields specific activities that are more than 22% of the soluble enzyme. The enzyme electrode can be stored in dry state for more than three months without any loss of activity. The biosensor was applied to the determination of several phenols and o-diphenols. The lowest detect limit is 0.02 mu mol/1 and the linear range was 1.0 X 10(-7)-1.0 X 10(-4) mol/1 for catechol. The kinetic parameters have also been calculated.

CONSTRUCTION OF A TYROSINASE-BASED BIOSENSOR IN PURE ORGANIC-PHASE

A novel immobilization method for construction of a tyrosinase-based biosensor applied in pure organic phase is described. This method gives the enzyme a hydrated shell which allows the enzyme to maintain its biocatalytic activity in a pure organic solvent The enzyme electrode was used to determine several phenols and o-diphenols in pure chloroform and chlorobenzene. The biosensor can be stored in dry state for more than 3 months without any loss of the activity. The kinetic parameters have also been calculated and are presented herein.

Electrochemical biosensor based on microfabricated electrode arrays for life sciences applications

In developing a biosensor, the utmost important aspects that need to be emphasized are the specificity and selectivity of the transducer. These two vital prerequisites are of paramount in ensuring a robust and reliable biosensor. Improvements in electrochemical sensors can be achieved by using microelectrodes and to modify the electrode surface (using chemical or biological recognition layers to improve the sensitivity and selectivity). The fabrication and characterisations of silicon-based and glass-based gold microelectrode arrays with various geometries (band and disc) and dimension (ranging from 10 μm-100 nm) were reported. It was found that silicon-based transducers of 10 μm gold microelectrode array exhibited the most stable and reproducible electrochemical measurements hence this dimension was selected for further study. Chemical electrodeposition on both 10 μm microband and microdisc were found viable by electro-assisted self-assembled sol-gel silica film and nanoporous-gold electrodeposition respectively. The fabrication and characterisations of on-chip electrochemical cell was also reported with a fixed diameter/width dimension and interspacing variation. With this regard, the 10 μm microelectrode array with interspacing distance of 100 μm exhibited the best electrochemical response. Surface functionalisations on single chip of planar gold macroelectrodes were also studied for the immobilisation of histidine-tagged protein and antibody. Imaging techniques such as atomic force microscopy, fluorescent microscopy or scanning electron microscope were employed to complement the electrochemical characterisations. The long-chain thiol of self-assembled monolayer with NTA-metal ligand coordination was selected for the histidine-tagged protein while silanisation technique was selected for the antibody immobilisation. The final part of the thesis described the development of a T-2 labelless immunosensor using impedimetric approach. Good antibody calibration curve was obtained for both 10 μm microband and 10 μm microdisc array. For the establishment of the T-2/HT-2 toxin calibration curve, it was found that larger microdisc array dimension was required to produce better calibration curve. The calibration curves established in buffer solution show that the microelectrode arrays were sensitive and able to detect levels of T-2/HT-2 toxin as low as 25 ppb (25 μg kg-1) with a limit of quantitation of 4.89 ppb for a 10 μm microband array and 1.53 ppb for the 40 μm microdisc array.