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.

Fibre-optic biosensor based on luminescence and immobilized enzymes: microdetermination of sorbitol, ethanol and oxaloacetate.

We have investigated highly selective and ultrasensitive biosensors based on luminescent enzyme systems linked to optical transducers. A fibre-optic sensor with immobilized enzymes was designed; the solid-phase bioreagent was maintained in close contact contact with the tip of a glass fibre bundle connected to the photomultiplier tube of a luminometer. A bacterial luminescence fibre-optic sensor was used for the microdetermination of NADH. Various NAD(P)-dependent enzymes, sorbitol dehydrogenase, alcohol dehydrogenase and malate dehydrogenase, were co-immobilized on preactivated polyamide membranes with the bacterial system and used for the microdetermination of sorbitol, ethanol and oxaloacetate at the nanomolar level with a good precision.

Design of luminescence photobiosensors.

The potential of immobilized enzyme membranes in biosensors has been explored in our group for several years. Although part of our work has been mainly devoted to electrochemical transducers and oxidases for the design of enzyme electrodes, the demand for ultrasensitive and highly selective sensors led us to consider the use of luminescent enzyme systems associated to optical transduction. When considering the need for operational and reliable biosensors in biotechnology, immobilization and stability of the sensing element still remain, in most cases, an unavoidable problem. We recently proposed a very fast and reliable procedure for preparing enzymatic membranes from Pall (Biodyne Immunoaffinity membranes) supplied in a pre-activated form. Both the firefly and bacterial systems as well as peroxidase for the chemiluminescent determination of various analytes, could be bound to such a support. Based on this approach, a fibre-optic sensor with immobilized enzymes has been designed which permits bio- or chemiluminescent analysis of ATP, NADH or H2O2 respectively. With the NADH-based system, other analytes could be detected using coupled dehydrogenases. This device appears very promising and includes the convenience of both the luminescence sensitivity as well as the handling of the biosensor design.

Optically-based chemical and biochemical sensors for the detection of some drugs and biological compounds.

The development of new methods for determining at a very low level a large spectrum of substances affecting the behaviour of living organisms is still a challenging goal. For such a purpose, chemical sensors which can be defined as the intimate combination of a sensitive and specific layer with a transducer, are undoubtedly among the more promising devices. In this field, optical sensors are expanding rapidly, mainly based on absorption, fluorescence, chemi- and bioluminescence. Beside pH and gases, drugs (anticonvulsant, antitumour, anaesthetic...) and other compounds of biological interest can be determined with specifically designed optical sensors, for instance immunosensors. Special attention will be given to optical biosensors with emphasis on chemi- and bioluminescence-based devices which are highly selective and ultrasensitive. When co-immobilizing various auxiliary enzymes in the sensing layer, the potentialities of such devices can be greatly extended as demonstrated by promising results recently obtained in our group.

Fibre-optic sensor with co-immobilised bacterial bioluminescence enzymes.

A fibre-optic bioluminescent sensor for the microdetermination of NADH is described. Measurements can be performed in the linear range 1 x 10(-9) M-3 x 10(-6) M with a detection limit of 3 x 10(-10) M using the bacterial luciferase and NAD(P)H:FMN oxidoreductase co-immobilised on a preactivated polyamide membrane. The relative standard deviation was 4.8-5.5% at 4 x 10(-8) M NADH and the steady-state response time was 2 min. When stored at -20 degrees C with 20% glycerol, the activity of the bioactive membranes was higher than that measured prior to freezing and then remained stable for more than four months.