Layer-By-Layer Assembled Enzyme Cascade for Catalyzing Oxidation of Sucrose for Biofuel Cells

Abstract

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.