Glucose oxidase (GOx)-based electrodes off er promising applications in glucose sensing and as potential power sources for

implantable devices, yet their performance remains critically dependent on effi cient electron transfer and enzyme immobilization

strategies. This study systematically investigated the co-immobilization of GOx and a redox-active osmium polymer,

poly (N-vinylimidazole)-[Os(4,4′-dimethyl-2,2′-bipyridine) 2 Cl]) +/2+ (PVI-Os-dme), using poly(ethylene glycol) diglycidyl

ether (PEGDGE) as a crosslinker to enhance both the catalytic and electron-transfer properties of the electrode. By varying

the enzyme-to-mediator ratio and applying a layer-by-layer assembly approach, we demonstrated that both loading quantity

and composition critically infl uenced current generation, charge transfer resistance, and overall electrode effi ciency. While

current output increased with additional layers, the catalytic activity per unit mass of enzyme or mediator decreased, indicating

a trade-off at high loadings. The optimized electrode, composed of six composite layers (2 μg GOx, 3.6 μg PVI-Os-dme,

2.2 μg PEGDGE per layer), achieved the highest peak current of 23.7 ± 1.7 μA at 0.3 V and retained over 85% of initial

current after 3 cycles and 57% after 5 cycles, demonstrating favorable reusability. Kinetic analysis revealed an apparent

Michaelis–Menten constant ( K m

app ) of 9.0 mM and a maximum current ( I max ) of 29.2 μA, confi rming the electrode’s high

affi nity and catalytic effi ciency toward glucose. These results highlight the importance of optimizing GOx/PVI-Os-dme

loadings, ratio, and the number of layers for enhancing the electrode performance.