In general there are numerous combinations of enzymes and mediators that have been employed in biofuel cells but the respective studies typically involve monoenzymatic systems, which are capable of only partial oxidation of the fuel. Improvement of fuel
utilization can be achieved by complete oxidation, which can be realized by introduction of enzyme cascades to increase the overall efficiency of the fuel cell (Nick et al., 2005; Sokic — Lazic et al., 2010; Addo et al., 2010). By this way, the overall performance of the BFC is increased as well.

In a theoretical work described by Kjeang and co-workers, a concept of an enzymatic fuel cell is proofed, without presenting experimental system (Kjeang et al., 2006). This work describes how to optimize the structure of a microfluidic enzymatic fuel cell, involving three-step-catalyzed methanol oxidation (Fig. 9). Different enzyme patterning strategies are tested, e. g., spatially distributed — or evenly-mixed enzymes along the electrode surface. The model predicts high fuel utilization at low flow rates, i. e., in the diffusion dominated and mixed mass, transfer conditions. According to the model, the investigated theoretical concept is reaction limited, which means that the system performance can be improved by improving enzyme turnover numbers. This work also demonstrates that the power required for pumping of the fuel is negligible in comparison to predicted power of the fuel cell.

Based on this work, we built a methanol/O2 microfluidic BFC able to completely oxidize methanol, according to the electron steps described for each electrode in Fig. 10. The enzymes and the mediators were immobilized in poly-L-lysine.

These enzymes are NADH-dependant. The electrochemical connection and regeneration of NADH at the anode is achieved using the enzyme diaphorase and the redox mediator benzylviologen as already described (Palmore et al., 1998). At the anode, a mixture of the three dehydrogenase enzymes was deposited along a gold electrode surface according two configurations (Fig. 11). When enzymes are mixed along the anode, optimal power density is achieved as observed in Fig. 12. The Y-channel device delivers a power density of 70 pW cm-2 at a cell voltage 0.25 V.

(B)

2. Conclusion and perspectives

Microfluidic BFCs could be an effective solution for small power sources applications such as biological sensors, implantable medical devices or portable electronics. However significant research efforts must be made for practical applications. Researches must be
aimed at identifying most robust and active enzymes, more efficient immobilization environment for enzymes and mediators in microfluidic environment, and at increasing enzyme lifetimes.

To deliver higher power densities, the challenges are in the area of energy density and fuel utilization. Microfluidic BFCs designs should integrate advanced immobilization configurations to improve enzyme performance and high surface area electrodes to enhance rates of convective-diffusive reactant transport. More nanofluidic research and development will be needed to demonstrate the real potential of this form of energy conversion system. Besides, the power output of single microfluidic BFC is inadequate for most practical applications. The enlargement of a single cell by increasing the geometrical electrodes area and the microchannel is constrained by fuel/oxidant crossover and higher ohmic losses. In order to produce adequate power, multiple independent cells could be stacked as in typical fuel cells.

3. Acknowledgment

This work was supported by a CNRS postdoctoral fellowship and by the Project PIE CNRS "Energie" 2010-2013. Nanolyon clean room facilities are acknowledged for the fabrication of the microfluidic devices.