Sonochemically fabricated microelectrode arrays for biosensors--part II. Modification with a polysiloxane coating.

A polymer-modified sonochemically fabricated glucose oxidase microelectrode array with microelectrode population densities of up to 2.5 x 10(5) microelectrodes per square centimetres is reported. These microelectrode sensors were formed by first depositing an insulating film on commercial screen printed electrodes which was subsequently sonicated to form cavities of regular sizes in the film. Electropolymerisation of aniline at the microelectrode cavities formed polyaniline protrusions containing entrapped glucose oxidase. Chemical deposition of polysiloxane from dichlorodimethysilane was used to deposit a thin protective and diffusion mass transport controlling coating over the electrodes. The physical and electrochemical properties of these films were studied. The performance of the final glucose oxidase based microelectrode sensor array is reported.

Ultra-thin-polysiloxane-film-composite membranes for the optimisation of amperometric oxidase enzyme electrodes.

An outer ultra-thin-polydimethyldichlorosiloxane film composite membrane has been used as the outer covering barrier in an amperometric glucose oxidase enzyme electrode biosensor. The composite membrane was formed via the condensation polymerisation of dimethyldichlorosilane at the surface of a host porous alumina membrane. Homogeneous polydimethyldichlorosiloxane films of <100 nm thickness acted as effective substrate diffusional barriers and were supported by the underlying porous alumina surface. Glucose and oxygen permeability coefficients were determined using diffusion chamber apparatus. Polysiloxane composite membranes were found to offer some screening functionality towards anionic biological interferents such as ascorbate. On exposure to blood an approximate 25% signal drift was observed during the first 2 h exposure to blood; after this time responses remained almost stable. Whole blood glucose determinations showed a close correlation (r(2)=0.98) to analyses performed via standard hospital analyses.

Polymer modified electrodes for the reversible oxidation-reduction of NAD+/NADH for use within amperometric biosensors.

Electrochemical enzyme based biosensors continue to attract much attention due to the simplified analyses they promise, in comparison to many more complicated analytical procedures. The largest group of enzymes, are those that rely NAD(P)+/NAD(P)H as cofactors. One of the problems associated with the use NAD(P)+/NAD(P)H redox couples, however, within electrochemical biosensors is their ability to passivate noble metal electrodes. There have been many attempts to overcome this problem, such as the use of carbon paste electrodes [1], although approaches such as these are both cumbersome and typically irreproducible. In recent years conducting polymers such as poly(3-methylthiophene) [2] have also been used to modify noble metal electrode surfaces to lower the overpotentials required, while poly(phenol red) has been incorporated into films to offer selectivity against interferents [3,4]. In this study platinum electrodes have been electrochemically coated with mixed polymer films of poly(phenol red) together with poly(3-methylthiophene), poly(aniline) or poly(o-phenylenediamine) to offer enhanced performance. Cyclic voltammetry has been used to compare the electrochemical behaviour of the NAD+/NADH redox couple over repeated potential cycles, with the most favourable results being seen with the poly(aniline)/poly(phenol red) and poly(o-phenylenediamine)/poly(phenol red) coated electrodes.

A simplified squalene epoxidase assay based on an HPLC separation and time-dependent UV/visible determination of squalene.

A novel and highly simplified enzyme assay for squalene epoxidase (EC 1.14.99.7) has been developed. The assay relies on the UV/visible determination of squalene at 195 nm, as it elutes from an octadecylsilane HPLC column. An acetonitrile/water (95.5/0.5, v/v) mixture was found to provide an ideal mobile phase, into which aqueous enzyme reaction mixture aliquots could be injected. Squalene, the natural substrate for squalene epoxidase, may be quantitatively determined within the concentration range 0-30 microM, with a calibration curve exhibiting an r2 (where r2 is the square of the Pearson correlation coefficient r) of 0.995. The HPLC retention time for squalene was significantly longer (> 15 min) than that for any other component required to prepare an enzyme assay reaction mixture, so facilitating its identification and quantification. In this way HPLC was used to follow enzymic squalene consumption within aliquots taken over a 30-min period. Previously reported squalene epoxidase assays rely on the radiolabeling and subsequent monitoring of squalene as it is metabolized by the enzyme. A highly simplified enzyme assay for squalene epoxidase is therefore reported.

The characterization of liposomal glucose oxidase electrodes for the measurement of glucose.

Many biosensors have been described for the measurement of glucose in order to monitor diabetic patients. Glucose oxidase has been used commonly in the construction of glucose sensors but the performance of this enzyme is limited by enzyme saturation kinetics, which restrict the measurement of clinically relevant glucose concentrations (0 to 25 mM). Diffusion limiting membranes have been described that result in the exposure of the enzyme to lower concentrations of glucose than are present in the bulk test solution. Recently a liposomal enzyme electrode was reported whereby glucose oxidase was encapsulated within liposomes so that the lipid bilayer was the diffusion limiting membrane. It was shown that the electrode response was defined by the lipid constituents of the liposome, and that a linear response to glucose could be achieved up to 40 mM. This paper describes research undertaken to improve the methods of production of a liposomal enzyme electrode. Improved immobilization of liposomes is demonstrated with the use of poly-L-lysine solution. The variation in electrode response with respect to the amount of glucose oxidase liposomally encapsulated is reported. The new method allows a greater number of sensors to be produced from a single batch of liposomes. Studies also show the biofouling effects of the lipid constituents of ruptured liposomes on the response of the electrode to glucose over time.

Biosensors: a viable monitoring technology?

Biosensors for practical in vivo and in vitro applications are dependent on the effective integration of several biological and physical technologies. This review paper was stimulated by an IEE seminar. Some of the more recent advances aimed at taking techniques of fundamental and academic interest to various forms of practical reagentless biochemical analysis are highlighted, with associated clinical and commercial consequences. The paper describes some of the most recent developments in biosensor research, in particular those relating to material aspects of fabrication, including multilayer films for sensor applications, advances in ISFETs, conjugated polymers, new developments in quartz crystal based biosensors, as well as advances in amperometric enzyme electrodes and the application of devices for continuous monitoring.

Selective membranes for the construction and optimization of an amperometric oxalate enzyme electrode.

Oxalate oxidase has been immobilized within a membrane laminate. Integration of the laminate within an amperometric electrode afforded a sensor for determining oxalate via electrochemical oxidation of H2O2 and subsequent current generation. A study of novel poly(vinyl chloride) (PVC) and cellulose acetate (CA) membranes with various outer/inner membrane configurations made it possible to eliminate direct electrochemical interference from species present in urine, typically ascorbate (60-500 mumol l-1), homovanillic acid (40-60 mumol l-1) and oxalate itself (50-500 mumol l-1). Of the membranes studied, only CA and plasticized PVC were found to screen out the direct oxalate electrochemical signal effectively. Cellulose acetate exhibited the best selectivity ratio for H2O2 over urine interference. An outer 0.05 micron pore radius polycarbonate membrane imparted sensor linearity in the narrow range 2-200 mumol l-1 oxalate, which fell below the normal range of oxalate in urine. The application of unplasticized PVC as an outer membrane allowed the sensor to exhibit linear characteristics which extended beyond the clinically relevant range (> 700 mumol l-1). The Michaelis-Menten constant of the immobilized enzyme was measured and found to be 10(-3) mol l-1.