Glucose and Glutamate Detection by Oxidase/Hemin Peroxidase Mimic Cascades Assembled on Macro- and Microelectrodes

Enzymatic cascades are routinely used for electroanalysis of redox inactive species that can be enzymatically converted into species electroactive at moderate potentials, such as H2O2 produced by FAD-dependent oxidases oxidising their substrates by O-2. However, such cascades adaptation to micro-biosensors is limited by enhanced mass-transfer of produced H2O2 into solution, not to the sensing layer. Here, bi-enzyme sensors for glucose or glutamate, produced by cross-linking peroxidase and oxidases on carbon-nanotube-modified graphite macro-electrodes (Gr), showed the from 0.1 to 10 mM glucose/glutamate linear response, while bio-modified 5 mu m carbon fibre electrodes (CFE) were mute due to fast transfer of enzymatically produced H2O2 into solution. By replacing peroxidase with peroxidase-mimicking hemin in polyethyleneimine, the sensitivity of detection at Gr improved 3-fold, enabling 2.8 mu M glucose and 4.5 mu M glutamate limits of detection, but not at CFE. Fast mass-transfer of H2O2 from CFE to solution was restricted by the Nafion membrane facilitating glucose detection at CFE with a sensitivity of 1.67 A cm(-2) M-1, at -0.6 V, escaping interference from other brain-fluid components, redox-inactive at this potential. Such membrane-restricted cascade system provides the analytical access to a large group of enzymes that can be integrated in multiple enzymatic cascades for biosensing at microelectrodes.