A water-based carbon screen-printing ink formulation, containing the redox mediator cobalt phthalocyanine (CoPC) and the enzyme glucose oxidase (GOx), was investigated for its suitability to fabricate glucose microbiosensors in a 96-well microplate format: (1) the biosensor ink was dip-coated onto a platinum (Pt) wire electrode, leading to satisfactory amperometric performance; (2) the ink was deposited onto the surface of a series of Pt microelectrodes (10-500 μm diameter) fabricated on a silicon substrate using MEMS (microelectromechanical systems) microfabrication techniques: capillary deposition proved to be successful; a Pt microdisc electrode of ≥100 μm was required for optimum biosensor performance; (3) MEMS processing was used to fabricate suitably sized metal (Pt) tracks and pads onto a silicon 96 well format base chip, and the glucose biosensor ink was screen-printed onto these pads to create glucose microbiosensors. When formed into microwells, using a 340 μl volume of buffer, the microbiosensors produced steady-state amperometric responses which showed linearity up to 5 mM glucose (CV=6% for n=5 biosensors). When coated, using an optimised protocol, with collagen in order to aid cell adhesion, the biosensors continued to show satisfactory performance in culture medium (linear range to 2 mM, dynamic range to 7 mM, CV=5.7% for n=4 biosensors). Finally, the operation of these collagen-coated microbiosensors, in 5-well 96-well format microwells, was tested using a 5-channel multipotentiostat. A relationship between amperometric response due to glucose, and cell number in the microwells, was observed. These results indicate that microphotolithography and screen-printing techniques can be combined successfully to produce microbiosensors capable of monitoring glucose metabolism in 96 well format cell cultures. The potential application areas for these microbiosensors are discussed.

A biosensor for the measurement of glucose in serum has been developed, based on a screen-printed carbon electrode modified with Meldola's Blue-Reinecke salt, coated with the enzyme glucose dehydrogenase (from Bacillus sp.), and nicotinamide adenine dinucleotide coenzyme (NAD+). A cellulose acetate layer was deposited on top of the device to act as a permselective membrane. The biosensor was incorporated into a commercially available, thin-layer, amperometric flow cell operated at a potential of only +0.05 V versus Ag/AgCl. The mobile phase consisted of 0.2 M phosphate buffer (pH 7.0) containing 0.1 M potassium chloride solution, and a flow rate of 0.8 ml min(-1) was used throughout the investigation. The biosensor response was linear over the range of 0.075-30 mM glucose, with the former representing the detection limit. The precision of the system was determined by carrying out 20 repeat injections of a 5-mM glucose standard, and the calculated coefficient of variation was 3.9%. It was demonstrated that this biosensor system could be applied to the direct measurement of glucose in serum without pretreatment. Therefore, this would allow high-throughput analysis, at low cost, for this clinically important analyte.

Microband glucose biosensors were fabricated by screen-printing a water-based carbon ink formulation containing cobalt phthalocyanine redox mediator and glucose oxidase (GOD) enzyme, then insulating and sectioning through the thick (20μm) film to expose a 3mm-long working electrode edge. The performance of these biosensors for glucose analysis was investigated at 25°C. Voltammetry in glucose-containing buffer solutions established that an operating potential of +0.4V vs. Ag/AgCl was suitable for analysis under both stirring and quiescent conditions. The influence of pH on biosensor performance was established and an operational pH of 8.0 was selected. Steady-state responses were obtained under quiescent conditions, suggesting a mixed mechanism predominated by radial diffusion, indicative of microelectrode behaviour. Calibration studies obtained with these biosensors showed steady-state currents that were linearly dependent on glucose concentration from the limit of detection (0.27mM) up to 2.0mM, with a precision for replicate biosensors of 6.2–10.7%. When applied to the determination of glucose in human serum, the concentration compared favourably to that determined by a spectroscopic method. These results have demonstrated a simple means of fabricating biosensors for glucose measurement and determination in situations where low-current real-time monitoring under quiescent conditions would be desirable.

The present study demonstrated for the first time that screen-printed carbon microband electrodes fabricated from water-based ink can readily detect H(2)O(2) and that the same ink, with the addition of lactate oxidase, can be used to construct microband biosensors to measure lactate. These microband devices were fabricated by a simple cutting procedure using conventional sized screen-printed carbon electrodes (SPCEs) containing the electrocatalyst cobalt phthalocyanine (CoPC). These devices were characterised with H(2)O(2) using several electrochemical techniques. Cyclic voltammograms were found to be sigmoidal; a current density value of 4.2 mA cm(-2) was obtained. A scan rate study revealed that the mass transport mechanism was a mixture of radial and planar diffusion. However, a further amperometric study under quiescent and hydrodynamic conditions indicated that radial diffusion predominated. A chronoamperometric study indicated that steady-state currents were obtained with these devices for a variety of H(2)O(2) concentrations and that the currents were proportional to the analyte concentration. Lactate microband biosensors were then fabricated by incorporating lactate oxidase into the water-based formulation prior to printing and then cutting as described. Voltammograms demonstrated that lactate oxidase did not compromise the integrity of the electrode for H(2)O(2) detection. A potential of +400 mV was selected for a calibration study, which showed that lactate could be measured over a dynamic range of 1-10mM which was linear up to 6mM; a calculated lower limit of detection of 289 microM was ascertained. This study provides a platform for monitoring cell metabolism in-vitro by measuring lactate electrochemically via a microband biosensor.

Microband glucose biosensors were produced by insulating and sectioning through a screen-printed, water-based carbon electrode containing cobalt phthalocyanine redox mediator and glucose oxidase enzyme. Under quiescent conditions at 37°C, at an operating potential of +0.4V, they produced an amperometric response to glucose in buffer solutions with a sensitivity of 26.4nA/mM and a linear range of 0.45 to 9.0mM. An optimal pH value of 8.5 was obtained under these conditions, and a value for activation energy of 40.55kJmol−1 was calculated. In culture medium (pH 7.3), a sensitivity of 13nA/mM was obtained and the response was linear up to 5mM with a detection limit of 0.5mM. The working concentration was up to 20mM glucose with a precision of 11.3% for replicate biosensors (n=4). The microband biosensors were applied to determine end-point glucose concentrations in culture medium by monitoring steady-state current responses 400s after transfer of the biosensors into different sample solutions. In conjunction with cultures of HepG2 (human Caucasian hepatocyte carcinoma) cells, current responses obtained in 24-h supernatants showed an inverse correlation (R2=0.98) with cell number, indicating that the biosensors were applicable for monitoring glucose metabolism by cells and of quantifying cell number. Glucose concentrations determined using the biosensor assay were in good agreement, for concentrations up to 20mM, with those determined spectrophotometrically (R2=0.99). This method of end-point glucose determination was used to provide an estimated rate of glucose uptake for HepG2 cells of 7.9nmol/(106 cellsmin) based on a 24-h period in culture.

A new method of using screen-printed carbon electrodes (SPCEs) incorporating the electrocatalyst cobalt phthalocyanine (CoPC) for the manufacture of tubular microband electrodes for hydrogen peroxide detection is described. Characterisation of these electrodes using potassium ferrocyanide, with cyclic voltammetry, has shown that steady state behaviour is displayed which is indicative of microelectrode behaviour. The current density obtained from the voltammogram was compared to that obtained for a conventional sized CoPC–SPCE, and the values were 5618 and 35.65μAcm−2, respectively. Cyclic voltammetry was carried out for the same electrodes, using 7mM H2O2 prepared in phosphate buffer at scan rates between 1 and 50mVs−1 and no significant increase in current response was observed. The application of these tubular microband CoPC–SPCEs, to the measurement of H2O2 using chronoamperometry was investigated. A calibration study was performed and the plot showed a sensitivity value of 252μAmM−1cm−2 and a lower detection limit of 70μM. We have shown that the chronoamperometric current response could be calculated using a modified equation originally developed for a plain microband electrode. This study provides a platform for using screen-printed carbon electrodes for the fabrication of oxidase based microbiosensors, for the determination of a variety of cellular metabolites.

A disposable biosensor for ammonium ions in sewage effluent was developed and based on a screen-printed carbon electrode coated with the enzyme glutamate dehydrogenase, 2-oxoglutarate, and NADH. This rapid and selective amperometric biosensor is capable of detecting NH4+ in sewage effluent at concentrations in the range 1-10 ppm. The biosensor was developed as a disposable, reagentless device incorporated into a fully automated, hand-held instrument that can be used away from the laboratory. This low cost, commercially available system permits the measurement of NH4+ in effluent samples in only 5 min and 20 s without any pretreatment. The results obtained correlated well with the standard laboratory method, used by the Environment Agency.

Screen-printed carbon electrodes (SPCEs) bearing a surface-adsorbed antibody against aflatoxin B 1 (AFB 1) were used in a competitive immunoassay, based on competition of free analyte with a biotinylated aflatoxin B 1 conjugate. Subsequent addition of streptavidin-alkaline phosphatase (AP) conjugate, followed by a 1-naphthyl phosphate substrate resulted in the production of the electrochemically active product, 1-naphthol; this was oxidized using linear sweep voltammetry and constituted the measurement step. Investigations were carried out to test the suitability of reagents by enzyme-linked immunoassay (ELISA) and to deduce the optimum blocking agent for the immunosensors. Using the electrochemical immunosensor, a calibration plot for AFB 1 was obtained over the concentration range 0.15 to 2.5 ng/mL, giving a detection limit of around 0.15 ng/mL in buffer solution. The immunosensors were fabricated in an array configuration, suitable for use in conjunction with 96-well microtiter plates; indeed, each step of the immunoassay was performed by dipping the electrodes into microwells. These results represent the initial studies towards the development of an automated instrument for multi-analyte determinations using immunosensor arrays for mycotoxin detection and form the basis for further studies.