Gold Nanoclusters with Two Sets of Embedded Enzyme Nanoparticles for Applications as Electrochemical Sensors for Glucose

Enzyme-based electrochemical biosensors have been widely used because of their sensitivity, rapidity, and highly target-specific reactions. However, the immobilized enzymes can hinder electron transfer on the surface of the electrode and can decrease the sensitivity of electrochemical biosensors. To overcome this problem, we synthesized conductive gold nanoclusters with two sets of embedded enzyme nanoparticles, which are called gold nanocluster-embedded dual-enzyme nanoparticles (AuNC-DENPs) composed of a glucose oxidase-stabilized gold nanocluster (GOX-AuNC) and a horseradish peroxide-stabilized gold nanocluster (HRP-AuNC). Moreover, the application of these nanoparticles as an enzyme-based highly sensitive electrochemical sensor was investigated. Owing to the effect of AuNCs, these nanoparticles have good conductivity compared to bare protein nanoparticles. In addition, the synthesized AuNC-DENPs enabled the combination of a two-enzyme cascade reaction, in which the GOX-AuNC component of the nanoparticle oxidized glucose to generate hydrogen peroxide, which then reacted with the adjacent HRP-AuNC component on the nanoparticle. Given the proximity of the two enzyme components in a single nanoparticle, the AuNC-DENPs markedly reduced the diffusion and decomposition of H2O2 during the cascade reaction and showed an enhanced catalytic reaction compared to a mixture of enzymes. As a result, the biosensor exhibited high sensitivity (18,944 μA/mM cm2) and could detect very low concentrations of glucose ranging from 5 to 320 nM and a LOD of 2.58 nM. In addition, by analyzing nontarget materials and serum together with glucose, it was confirmed that the sensor has good selectivity for glucose.