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Optical biosensing techniques for monitoring organic pollutants in the aquatic environment

Exploitable results

Electrochemiluminescent immunoassays on indium tin oxide coated waveguides have been developed. Indium tin oxide coated glass was silanized by passing aminosilane vapour over the surface or by reflux in a solution of the aminosilane in dry toluene. Small fluorophores and large enzyme molecules can attach to the derivatized surface. The small fluorophore fluorescamine reacts rapidly and specifically with primary amines yielding a fluorescent product that can be measured in situ. Investigation of the 2 methods indicated that indium tin oxide silanized with aminosilane vapour had more amino groups on the surface. The enzyme horseradish peroxidase was attached with N-succinimidy 3-(2-pyridylthio)propionate (SPDP) or glutaraldehyde. The SPDP method gave more reliable results. Immobilized horseradish peroxidase can be used to generate a coloured dye and the amount of dye is a measure of the amount of enzyme attached to the surface. Results indicate that there is little difference between the 2 methods of silanizing. An electrochemiluminescent immunoassay has been developed based on the generation of light when luminol is oxidized at an indium tin oxide coated electrode in the presence of hydrogen peroxide. Hydrogen peroxide was generated using the enzyme glucose oxidase, which has been attached to antibodies specific for pesticides using glutaraldehyde. Ovalbumin labelled with coumarin has been attached to indium tin oxide coated glass using the same method as for horseradish peroxidase. Antibodies to coumarin were labelled with horseradish peroxidase and incubated with ovalbumin derivatized indium tin oxide coated glass. Comparison with underivatized blanks showed that the antibodies had bound to immobilized coumarin. Conditions for detecting the enzyme label are being optimized using glucose oxidase immobilized on indium tin oxide coated glass in a flow cell luminometer. The immobilization technique is identical to that used for horseradish peroxidase and ovalbumin. Pesticide immunoassays have been successfully carried out using the electrochemiluminescent detection technique developed.

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