Layer-by-Layer Assembled Enzyme Cascade Bioanode for Catalyzing Oxidation of Sucrose

Feasibility of layer-by-layer (LbL) assembly via electrostatic interaction in development of multi-enzyme based biosensors was successfully demonstrated in our previous research. In this study, application of LbL technique was extended for the first time in assembly of multi-enzyme, invertase (INV) and NAD+-dependent glucose dehydrogenase (GDH) for cascade catalytic oxidation of sucrose for biofuel cell application. The system was constructed through LbL assembly of electrostatically interacted INV and GDH armored multi-walled carbon nanotubes (MWCNT-INV and MWCNT-AChE) with a set of cushioning bilayers consisting of MWCNT-polyethyleneimine (PEI) and MWCNT-DNA on screen printed electrode (SPE). The LbL architecture shows great advantages for sequential enzymatic reaction that it favors the efficient penetration of substrate and products in a cascade system. The utilization of MWCNT facilitates the direct electron transfer from sucrose oxidation and enhances the current generated. The current density of 104.08±0.85 μA/cm² using Amperometry was currently achieved with 50 mM sucrose and 1 mM NAD⁺ on SPE with a surface area of 0.2 cm² in PB buffer 6.0 at room temperature. The current density of LbL assembled electrode can be further improved by optimization of buffer pH, concentration of NAD+ as well as sucrose concentration. Therefore, the results indicated that the versatile LbL assembly is a simple and efficient way to co-immobilize CNT-multi-enzyme for cascade system, showing great potential in enzymatic biofuel cell development.