Retaining enzymes in functionally-active forms on the electrode surface is a challenging and difficult task. Liquid crystalline phases formed by polar lipids in aqueous media are widely used as model matrices to mimic biological processes. The lipidic cubic phase can be characterized as a curved bilayer forming a three-dimensional crystallographically well-ordered structure that is interwoven by aqueous channels. It provides a stable, well-organized environment in which diffusion of both water-soluble and lipid-soluble compounds can take place. The phases are isotropic and optically transparent and, therefore, they are ideal matrices also for spectroscopic investigations by which the functionality of any active site present in the matrix can be probed. Small solutes and water-soluble proteins diffuse within the network of aqueous channels allowing crystallization from this compartment.
Cubic phases based on monoacylglyceroles form readily and have attracted large interest due to their ability to incorporate and stabilize membrane proteins. Their lyotropic and thermotropic phase behaviour have been thoroughly investigated. Phase diagram of monoolein (monooleoyl-rac-glycerol, MO), water mixtures will be described. At hydration over 20% lipidic cubic phases Ia3d and Pn3m are formed. The latter are stable in the presence of excess of water. The cubic phase is a gel-like material which upon introduction of some compounds or large concentrations of supporting electrolyte, may undergo phase transition, so the phase should be monitored, preferably by polarized light microscope. Deterioration of cubic phase is reflected by turbidity. Cubic phases Pn3m can be stored for days without any phase transitions even at temperatures as low as 5oC. Due to high viscosity and stability in the presence of water these cubic phases can be simply smeared over solid substrates such as electrodes and used to host enzymes and synthetic catalysts leading to new types of catalytically active modified electrodes. We have shown earlier that cubic phase modified electrodes are useful for the determination of cholesterol and CO2 (Ropers et al. 2001, Rowiński et al. 2002, 2003).
In order to describe the efficiency of transport of small hydrophilic molecules within the film we determined loading times, diffusion coefficients and concentrations of selected redox mediators in the layer by voltammetry and chronocoulometry. Two types of electrodes were used: a normal size electrode working in the linear diffusion regime and an ultramicroelectrode working under spherical diffusion conditions. This allowed to determine together concentration and the diffusion coefficient in the medium in direct contact with the electrode surface
The monoolein-based cubic phase matrix was used for immobilizing laccases from Trametes sp. and Rhus vernicifera on the electrode surface. The contact between the electrode and the enzyme was maintained using suitable electroactive probes. Using the probe in its oxidized form allowed turning the reaction on or off, by applying an adequate potential to the electrode and generating the probe in the form undergoing the enzymatic transformation. The electrodes modified with cubic phases containing laccase and the hydroquinone/quinone couple were used for reducing dioxygen in aqueous solutions.
References
1. M.-H. Ropers, R. Bilewicz, M.-J. Stebe, A. Hamidic, A. Micloc, E. Rogalska, PhysChemChemPhys., 3 (2001) 240.
2. P. Rowinski, R. Bilewicz, M. J. Stebe, E. Rogalska, Anal. Chem., 74 (2002) 1554.
3. P. Rowinski, A. Korytkowska, R. Bilewicz, Chem. Phys. Lipids., 124 (2003) 147.
|