Layer-By-Layer Assembled Carbon Nanotube Immobilized Enzyme Cascade for Sucrose/O2 Biofuel Cell Utilization

A layer by layer (LbL) assemble technique for the first time was employed to modify glassy carbon electrode (GCE) and screen printed electrode (SPE) utilizing multiwalled carbon nanotube (MWCNTs) based polyelectrolytes and enzyme cascade to facilitate efficient electron transfer in Sucrose/O₂ biofuel cell. Previously, our group demonstrated layer-by-layer (LbL) assembly in fabrication of carbon nanotube (CNT) immobilized multi-enzyme biosensors for discriminative detection of multiple analytes. In this study, electrostatically interacted CNT-Invertase (INV), CNT-glucose dehydrogenase (GDH, NAD+-dependent) with cushioning bilayers CNT-polyethyleneimine (PEI) and CNT-DNA were alternatively assembled in LbL fashion on SPE (0.2 cm²) and GCE (0.07065 cm²). [Ni(phendion)(phen)]Cl₂ complex and methylene green (MG) were investigated as redox mediator for electrocatalytic oxidation of NADH at reduced overpotentials. The LbL architecture showed advantages for sequential enzymatic reaction that favored the efficient penetration of substrate and products in a cascade system while MWCNT facilitated the direct electron transfer from sucrose oxidation and enhancement of the current density. With GCE as modified bioanode using MG and Ni complex, the biofuel cell produced a higher power density (μW/cm²) with 134.4% and 34.3% enhancement comparing with SPE bioanode, respectively. With Ni complex compared modified on GCE and SPE, the biofuel cell produced a higher power density (μW/cm²) with 16.6% and 101.9 % higher comparing with MG modified on GCE and SPE, respectively. The LbL assembly showed great feasibility as a simple and efficient way to construct controlled CNT-multi-enzyme modified electrodes for biofuel cell applications.