Symposium 1997: Abstract for ÂR Ribeiro et al

Symposium 1997: Abstracts

Immobilisation of Firefly Luciferase on Glass Strips as an Alternative Strategy for Luminescent Detection of ATP

Ângela R. Ribeiro¹, Célia M. Antunes¹, Luís M. Rosário^1,2and Maria H. Gil³
¹Center for Neuroscience and Cell Biology, University of Coimbra, Portugal
²Department of Biochemistry, FCTUC, P.O. Box 3126, 3000 Coimbra, Portugal
³Department of Chemical Engineering, FCTUC, Coimbra, Portugal

The bioluminescent reaction catalysed by firefly luciferase has become widely established as an outstanding analytical system for assay of ATP. When used in solution luciferase is unstable and cannot be reused, problems that can be partially circumvented by immobilising the enzyme on solid substrates. Transparent glass is especially advantageous over alternative immobilising matrices since it allows most of the emitted photons to be detected. We report an alternative method for luciferase immobilisation on glass which does not require prior silanization and glutaraldehyde activation, thus saving preparation time and minimizing enzyme inactivation.

Our method is based on the co-immobilisation by adsorption of luciferase (from a firefly lantern extract) and poly-L-lysine (PL) on non-porous glass strips. Luciferase immobilised in this way exhibits minimal variations in inter-sample activity, high sensitivity for ATP detection (linear luminescence responses down to 10 pmols) and fairly good stability (half-life at 4 ^oC, 23 days; full activity for at least 60 days when stored at -80 ^oC). A further validation of this non-covalent immobilisation method was achieved by assaying the ATP levels of pancreatic islets, after extraction with Triton X-100. PL-mediated immobilisation of luciferase on glass strips provides an attractive strategy for the design of specific ATP biosensors, with potential in industry, environmental screening, medicine and biological research.