MULTICOMPONENT ORGANIZED BIOACTIVE LAYERS FOR FIBEROPTIC LUMINESCENT SENSORS

The characteristics and performance of biosensors mainly depend on the properties of the bioactive layer associated with the transducer. Two approaches that we consider as essential are addressed. the designs of (i) a reagentless fiber-optic biosensor with cosubstrates embedded in the vicinity of the immobilized enzyme; (ii) a compartmentalized layer with sequential enzymatic reactions for improved signal detection. Concerning the reagentless biosensor, we focus on the bacterial bioluminescent system involving two enzymes, oxidoreductase and luciferase, for NADH detection. The difficulty is in the achievement of both the confinement in the same matrix and subsequent release of the two coreactants, FMN and decanal. Among the difficulties in designing a reliable and substainable sensor, the difference in the affinity for water of the coreactants, and the desirability of a high loading together with a slow release rate avoiding microenvironmental inhibition can be mentioned. When associating such a self-contained sensing layer with the transducer, the biosensor can be operated for 1.5 h without reloading. For the studies of compartmentalized enzyme layers, the sequential bienzymatic system lactate oxidase-peroxidase is chosen as a model. The hydrogen peroxide produced by the lactate oxidase reaction serves as a cosubstrate for the chemiluminescence reaction of luminol catalyzed by peroxidase. Compartmentalization of the bioactive layer is obtained by immobilizing the two enzymes separately on different membranes stacked at the sensing tip of the fiber-optic sensor. When using such a design, a 20-fold increase of the sensor response for lactate is obtained compared with a sensor including lactate oxidase and peroxidase randomly coimmobilized on the same membrane.