Design of bioluminescence-based fiber optic sensors for flow-injection analysis

A fiber optic sensor based on enzyme-catalyzed light-emitting reactions has been developed and integrated in a flow-injection analysis (FIA) system. The firefly luciferase, specific for ATP, and the bacterial oxidoreductase/luciferase system, specific for NADH, have been immobilized on preactivated polyamide membranes. ATP and NADH analysis could be performed in the range from 0.1 pmol to 3 nmol and from 0.5 pmol to 1 nmol, respectively. By co-immobilizing these two bioluminescence systems on the same membrane, a multi-function biosensor has been designed allowing the alternate determination of ATP or NADH with the same sensitivity as that obtained with the two different mono-functional biosensors. A partly self-contained biosensor has been also developed for the flow injection analysis of NADH. For this purpose, FMN (one of the substrates of the bacterial bienzymatic system) has been embedded in a synthetic matrix. Different supports have been tested for the non-covalent immobilization of this substrate and its release in the immediate vicinity of the bound enzymes. Using a photo-crosslinked poly(vinyl alcohol) support, 40 reliable assays (CV = 4.5%) could be performed without changing or reloading the matrix.