A few works to date present enzyme-based microfluidic BFCs with immobilized enzymes. Enzymes in solution are only stable for a few days, it is why immobilization of active enzymes is of interest in order to improve lifetime (Bullen et al., 2006) and to develop integrated microfluidic BFC designs. Besides, full selectivity of both enzymatic half-cells allows microfluidic BFC operation in a single, combined fuel and oxidant channel with mixed reactants at constant concentrations favourable for stability studies. Additionally, the close proximity of the enzymes with the electrodes reduces ohmic losses because electrons are harvest from reaction sites with lower electrical resistance.

In microfluidic BFCs described in literature, enzymes with their respective redox mediators are immobilized on electrodes surface by encapsulation in polymer film. Currently, Nafion® is commonly used as it possesses surfactant properties interesting to immobilize enzymes in micellar structures when treated by quaternary ammonium salt (Moore et al., 2004). Such matrix provides an optimal enzyme environment where the enzyme retains its activity for greater than 90 days. Based on this strategy, a microchip-based bioanode with alcohol dehydrogenase enzymes immobilized in treated Nafion® associated with an external platinum cathode delivered a power density of 5 gW cm-2 (Moore et al., 2005). The low value was attributed to the thick coating of polymer, casting by hydrodynamic flow and thus difficult to control in the microchannels.

Another polymer to entrap enzymes is poly-L-lysine. This polymer mixed with an enzyme solution can stand on electrode surface after drying in air. According to this technique, a microfluidic glucose/O2 BFC was developed (Togo et al., 2007; Togo et al., 2008). The originality of this study concerned the location of the electrodes in a single flow channel. This device has been developed to generate electric power from glucose oxidation with an anode coated by immobilized glucose deshydrogenase and a bilirubin oxidase-adsorbed O2 cathode (Togo et al., 2008). The device was featured with a specific electrode-arrangement within a microchannel to prevent dissolved O2 to react at the anode as an interfering substance. A large biocathode (10 times the anode size) was placed strategically upstream of a bioanode to pre-electrolyse O2 to protect the anode vicinity from interfering oxygen. The maximum cell current was increased by 10% with this cell configuration at pH 7. The experimental results showed the influence of the channel height that should be in the same order of the depletion layer thickness for optimal operating of the device. However, in such design, the composition of fuel and oxidant could not be adjusted independently for optimum enzymatic activity and stability.

In order to choose independently the composition of the two streams, the configuration based on Y-microfluidic single channel is required. This approach has been developed for a microfluidic glucose biofuel cell working from GOx and laccase immobilised on gold electrodes in a poly-L-lysine matrix. The immobilization process was realized by mixing the enzymes and their respective redox mediators in poly-L-lysine solution. After drying, the device was tested with a phosphate buffer pH 7 solution with 50 mM glucose for the anolyte, and with a citrate buffer pH 3 solution saturated with oxygen for the catholyte. Different redox mediators were tested for efficient electron transfer at the anode. Among the mediators hexacyanoferrate (Fe(CN)63-), ferrocene and 8-hydroxyquinoline-5-sulfonic acid hydrate (HQS), the higher power density (60 pW cm-2) was obtained at a cell voltage 0.25 V with Fe(CN)63- (Fig. 8).