Ibec Seminar. Anne de Poulpiquet
CHARACTERIZATION OF ENZYMATIC BIOELECTRODES BY IN SITU FLUORESCENCE MICROSCOPY
A. de Poulpiquet,1 A. Guessab, 1 H. M. Man,1 I. Mazurenko,1 L. Bouffier,2 E. Lojou1
1Aix-Marseille Univ., CNRS, Bioenergetics and Protein Engineering, UMR 7281, Marseille
2 Institute of Molecular Sciences, UMR CNRS 5255, Univ. Bordeaux, ENSMAC, Pessac
adepoulpiquet@imm.cnrs.fr
Redox enzymes present remarkable catalytic properties (exceptional selectivity, high kinetic constant, low overvoltage, etc.) which are particularly interesting for bio-electrochemical devices (biosensors, biofuel cells, bioreactors). In the latter, they are immobilized at the surface of an electrode to enable electron transfer. Using three-dimensional (3D) electrodes improves the performance of the devices (sensitivity, current densities). However, enzymatic catalysis is very sensitive to the local environment (pH, temperature, ionic strength, concentration of substrates, products or inhibitors, etc.) whose composition, in the case of interfacial reactions, can differ from the bulk of the solution. These disparities are exacerbated when the enzymes are confined in the pores of 3D electrodes, due to the complexity of the associated mass transport. However, electrochemistry only provides indirect information on the environment of the electrode. Therefore, there is a major interest in coupling electrochemical techniques to other methods for collecting simultaneously spatial information.1-3 Precious information about mass transport and reactivity can be obtained by investigating the concentration profiles of the different species near the electrode surface, or in the volume of a porous electrode. We show that in situ fluorescence confocal laser-scanning microscopy (FCLSM) coupled with electrochemistry enables investigation of redox enzyme reactivity involving the indirect generation of fluorogenic species.4, 5 One of the most interesting features of FCLSM is the possibility to reconstruct 3D concentration profiles. Recording fluorescence in the volume adjacent to the electrode under potential control thus enables rebuilding the diffusion layer.2-5 We show that the method can be implemented to characterize electro-enzymatic catalysis at various planar and structured 3D electrodes.4, 5 For example, enzymatic O2 reduction involves proton transfers, which was evidenced via the fluorescence change of strongly pH-dependent fluorophores. Local pH changes in the electrode plane were measured during O2 reduction catalyzed by an immobilized bilirubin oxidase. Moreover, proton gradients generated during the enzymatic electrode reaction were imaged and their expansion under various experimental conditions were determined. Finally, the method enabled direct imaging of the evolution of confined environments in porous 3D electrodes such as gas-diffusion layers during electro(enzymatic) catalysis.
