Stephen F. White

Lactate, glutamate and glutamine biosensors based on rhodinised carbon electrodes

Amperometric enzyme sensors for lactate, glutamate and glutamine were constructed using rhodinised carbon electrodes. All three sensors operated at a potential of + 400 mV (Ag/AgCl). Lactate sensors were constructed using lactate oxidase immobilized in hydroxyethylcellulose, overlaid with a cellulose acetate membrane. Using a flow-injection system, the sensors had a linear range of 0.1 to 1.5 mM. Measurements of lactate concentrations from a mammalian cell culture were compared with results obtained from a commercial instrument. A correlation coefficient of r = 0.982 (n = 15) was obtained. Glutamate and glutamine sensors were fabricated based on glutaraldehyde immobilization; the former by incorporating glutamate oxidase and the latter based on glutamate oxidase and glutaminase (both had a linear range of 0.1 to 1.5 mM). The feasibility of using these sensors, in conjunction with a previously described glucose sensor, for mammalian cell culture monitoring is discussed.

Development of a mass-producible glucose biosensor and flow-injection analysis system suitable for on-line monitoring during fermentations

Screen printed amperometric glucose biosensors were constructed and optimised for use in a flow injection analysis (FIA) system. The sensors were fabricated using a catalytic metallised carbon based ink, allowing a working potential of +350 mV (Ag/AgCl). Overall the sensors displayed a high degree of reliability and robustness. A linear response was obtained over the range 0.1 to 25 mM glucose with a slope of 1.35 ± 0.20 μA/mM. The within electrode coefficient of variation at each concentration tested (n = 10) was less than 2% (or 0.1 mM for concentrations below 5 mM). In addition, it was demonstrated that a single sensor can be used in the FIA system over a seven day period indicating a high level of operational stability. These sensors fulfil the criteria necessary for a mass produced device, which would be suitable for the on-line monitoring of glucose consumption during microbial fermentation.

Development of a common biosensor format for an enzyme based biosensor array to monitor fruit quality.

Individual enzyme-based biosensors involving three-electrode systems were developed for the detection of analytes comprising markers of the stage of maturity and quality in selected fruits of economic importance to tropical countries. Importantly, a common fabrication format has been developed to simplify manufacture and allow future integration of the individual sensors into a single multi-sensor array. Specifically, sensors for beta-D-glucose, total D-glucose, sucrose and ascorbic acid have been developed. Pectin, a natural polysaccharide present in plant cells, was used as a novel matrix to enhance enzyme entrapment and stabilisation in the sensors. Except for ascorbic acid, all the sensors function via the detection of enzymatically generated H2O2 at rhodinised carbon electrodes. Since ascorbic acid is electrochemically active at the working potential chosen (+350 mV vs. Ag/AgCl), it was measured directly. Enzyme sensors demonstrated expected response with respect to their substrates, typically 0-0.8 microA/20 mm2 electrode area response over analyte ranges of 0-7 mM. Interferences related to electrochemically active compounds present in fruits under study were significantly reduced by inclusion of a suitable cellulose acetate (CA) membrane or by enzymatic inactivation with ascorbate oxidase. Initial development was carried out into production of biosensor arrays. CA membranes were used to improve the linear range of the sensors, producing up to a fivefold improvement in the detection range compared to sensors without an additional diffusion barrier.

Measurement of protein using an electrochemical bi-enzyme sensor.

A screen-printed three-electrode amperometric biosensor for the rapid and quantitative measurement of single protein solutions is described. A membrane immobilised protease preparation of broad specificity was used to digest sample protein liberating free amino acids that were subsequently oxidised at a working electrode by immobilised L-amino acid oxidase (L-AAO). The enzymatically generated hydrogen peroxide was determined amperometrically. The fully optimised device required 30 mU L-AAO and 3.94 U protease and had a limit of detection of 170 microg ml(-1) and linearity of response up to 1 mg ml(-1) for Casilan 90 protein. The analytical performance of the device was comparable to that of a commercially available standard photometric protein test kit and required only a 10 microl volume of sample and a single dilution step. Unlike with photometry, the sensor is able to determine the protein content of turbid samples and hence should find widespread applications. The device was simple to use, low-cost and could be mass-produced, yielding results within 4 min of sample addition with acceptable assay repeatability.

Monitoring of the glucose concentration during microbial fermentation using a novel mass-producible biosensor suitable for on-line use

A flow-injection analysis system was combined with a mass producible, disposable biosensor and was used to monitor glucose concentrations during several microbial fermentations. The biosensor was m ...

On-line monitoring of glucose, glutamate and glutamine during mammalian cell cultivations

Amperometric biosensors (based on rhodinised carbon electrodes) for glucose, glutamine and glutamate were constructed. The sensors were incorporated into a three cell parallel FIA system and used to monitor the three analytes on-line during two mammalian cell perfusion cultures. All measurements were made simultaneously from undiluted media sample. Use of the FIA system enabled easy and rapid exchange of the sensors, during cultivation. The inclusion of a calibration step, regularly for all sensors, helped to maintain the accuracy of all measurements. Comparison with off-line measurements indicated that all three biosensors operated successfully, providing accurate information.