Steven John Setford

Screen-printed amperometric biosensors for the rapid measurement of L- and D-amino acids

Screen-printed three-electrode amperometric sensors incorporating L- and/or D-amino acid oxidase for the general purpose measurement of L- or D-amino acids is described. The working electrode incorporates rhodinized carbon, to facilitate hydrogen peroxide oxidation at a decreased operating potential, and immobilized enzyme. The devices responded to all 20 common L-amino acids and all of the D-amino acids examined, the exceptions being L- and D-proline. Linear response profiles were observed for L-leucine, L-glycine and L-phenylalanine with limits of detection of 0.47, 0.15 and 0.20 mM respectively. The devices were reproducible and exhibited stability over a 56 d test period. The biosensor compares favourably with a standard photometric amino acid test and was used to monitor milk ageing effects. The assay is cheap, simple to perform and rapid, requiring only buffer-electrolyte and a small sample volume.

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.

Immunosensing in organic and mixed aqueous–organic phase environments

Immunosensors are devices incorporating antibody-based recognition elements allied to a suitable transducer and offer a wide range of simple diagnostic tools with powerful analytical performance in the aqueous phase. The need to monitor poorly water-miscible compounds has resulted in increasing research into the behaviour of such devices in the organic phase.

Peroxidase enzyme sensor for on-line monitoring of disinfection processes in the food industry.

For desirable environmental reasons, peroxides have replaced halogenated substances for disinfection purposes in the food and beverage industry. However, cost issues and the requirement to remove these agents completely after disinfection necessitate simple, low-cost and sensitive test methods with a wide dynamic range and on-line capability. The development and performance of such a method is detailed here. Low-cost peroxide sensors were fabricated using a single deposition procedure, in which horseradish peroxidase enzyme and dimethylferrocene mediator were entrapped within a cellulose acetate membrane, over the working electrode area of a screen-printed three-electrode assembly. Optimum performance was obtained using HRP and DMFc loadings of 25 U and 0.03 micromol per electrode, respectively, and a mean cellulose acetate molecular weight of 37,000. The device had a detection limit of 49.5 microM hydrogen peroxide and mean RSD values of 21% across the concentration range 49.5-368 microM. In laboratory studies the sensor was shown to have a stability of > or = 4 d in continuous flow-mode maintaining an accuracy of +/- 16% that was considered acceptable for the intended on-line monitoring of the disinfection process. In a field study, it was successfully used on-line within a flow-cell to measure peroxide levels during disinfection of an industrial fermentation vessel.

Electrochemical assay method for the rapid determination of oxidase enzyme activities

The standard photometric method for the determination of oxidase enzyme activity is compared with an electro-chemical approach, involving the use of cheap, disposable screen-printed electrodes. Development and characterisation of the electrochemical device is reported. The electrochemical approach is demonstrated to be advantageous, particularly with regard to speed, ease-of-use, chemicals required and decentralisation of analysis.

L-malic acid biosensor for field-based evaluation of apple, potato and tomato horticultural produce.

A screen-printed three-electrode amperometric biosensor incorporating malic enzyme for the measurement of L-malic acid in apple, potato and tomato horticultural samples has been developed. The working electrode contained 0.38 mU of immobilised enzyme and was fabricated using rhodinised carbon to facilitate NADPH oxidation at an operating potential of +300 mV vs. Ag/AgCl compared with > +600 mV for bare carbon. The linear range of the sensor was 0.028-0.7 mM L-malic acid with relative standard deviations of 3.3-13.3%. When testing with real apple, potato and tomato samples, the sensor accuracy was within 13.7% of a standard commercially available photometric test kit. The sensor approach is cheap, simple to perform and rapid (6 min), requiring only buffer-electrolyte and a small sample volume.

Quantitative immunoassay for determining polyaromatic hydrocarbons in electrical insulating oils

Abstract The development and application of a combined sample extraction and immunoassay protocol for the quantification of polyaromatic hydrocarbons (PAHs) in transformer oils is reported. Tests were performed on 12 different used transformer oils from three major manufacturers. The removal of matrix interferents was achieved by loading oil fractions onto silica solid phase extraction cartridges and eluting with non-polar solvent prior to evaporation and reconstitution in a more polar medium. Extracts were immunoassayed using two commercially available PAH test kits either having broad specificity towards priority PAHs or enhanced binding specificity toward more carcinogenic PAHs. The total and carcinogenic PAH test kits yielded PAH levels in the oil extracts 5.86-fold and 126-fold lower than the industry-standard IP346 method. The latter method, widely used by the industry, since it correlates with biological carcinogenicity tests, grossly over-estimates PAH levels in oils since it is a non-specific gravimetric solvent extraction approach. The assay was found to be unaffected by the extract sample matrix and was capable of determining PAHs at the nanogram per millilitre level. The assay protocol was simple, low-cost and rapid (

Field-based supercritical fluid extraction and immunoassay for determination of PAHs in soils

A field-compatible supercritical-fluid extraction (SFE) device and method for extraction of organic contaminants from soil has been developed and combined with field-based immunoassay for on-site PAH (polycyclic aromatic hydrocarbon) determination. The optimised extraction method was tested in field experiments on natural samples with varying water content (0–32% w/w) without any sample pretreatment, yielding an average PAH recovery of 80% versus Soxhlet. The immunoassay functioned in buffer-diluted 10% v/v MeOH SFE extracts, allowing direct determination of PAHs with minimum sample manipulation. Immunoassay served as a reliable semi-quantitative technique for rapid screening of PAHs in SFE preparations of natural samples extracted in the field. Poor performance of the commercial solvent-shake extraction (SSE) method further supported the on-site SFE/immunoassay method.

Electrochemical sensor for predicting transformer overload by phenol measurement

Transformer overload is a significant problem to the power transmission industry, with severe safety and cost implications. Overload may be predicted by measuring phenol levels in the transformer-insulating oil, arising from the thermolytic degradation of phenol-formaldehyde resins. The development of two polyphenol oxidase (PPO) sensors, based on monitoring the enzymatic consumption of oxygen using an oxygen electrode, or reduction of enzymatically generated o-quinone at a screen-printed electrode (SPE), for the measurement of phenol in transformer oil is reported. Ex-service oils were prepared either by extraction into aqueous electrolyte-buffer, or by direct dilution in propan-2-ol, the latter method being more amenable to simple at-line operation. The oxygen electrode, with a sensitivity of 2.87 nA microg(-1) ml(-1), RSD of 7.0-19.9% and accuracy of +/-8.3% versus the industry standard International Electrotechnical Commission (IEC) method, proved superior to the SPE (sensitivity: 3.02 nA microg(-1) ml(-1); RSD: 8.9-18.3%; accuracy: +/-7.9%) and was considerably more accurate at low phenol concentrations. However, the SPE approach is more amenable to field-based usage for reasons of device simplicity. The method has potential as a rapid and simple screening tool for the at-site monitoring of phenol in transformer oils, thereby reducing incidences of transformer failure.

Measurement of native dextran synthesis and sedimentation properties by analytical ultracentrifugation

The principle of integrated enzymatic synthesis and product separation is examined with the aid of an analytical ultracentrifuge. The biosynthesis of high molecular weight dextran polymer from sucrose using dextransucrase enzyme was chosen as the test reaction. Tests on pre-synthesised native dextran served to evaluate the process whilst allowing assessment of native dextran sedimentation properties. Native dextrans were found to have fully corrected sedimentation coefficients of 143–531 Svedbergs at infinite dilution. The biocatalytic formation of dextran was observed both visually and by measurement of the build-up of sedimented material in the ultracentrifuge cell. Sedimentation coefficients comparable to those observed for pre-synthesised dextran were recorded. © 1999 Society of Chemical Industry