Scanning Electrochemical Impedance Microscopy-Based Assessment of Glucose Biosensors.

Scanning electrochemical impedance microscopy (SEIM) was assessed as an electrochemical method for developing glucose biosensors based on glucose oxidase (GOx). To determine the lowest detectable GOx activity, scanning electrochemical microscopy (SECM) in the feedback mode (FB-SECM) was applied. During the measurement procedure, an ultramicroelectrode (UME) was moved vertically over the surface modified by immobilized GOx. A positive feedback response of the FB-SECM mode was determined during the assessment of surfaces modified by 5 fg/mm2 to 20 μg/mm2 surface concentration of GOx. The lowest surface concentration of GOx, which still provided reliable measurement results, was 50 fg/mm2. The approach curves registered using the FB-SECM mode were assessed using a mathematical model adapted for the calculation of reaction kinetics by SECM. According to this model, the reaction kinetics constant λ was calculated for differently modified surfaces in the presence of the same glucose concentration. For the surface not modified by GOx, the constant λ was determined to be 0.14, while for the GOx-modified surface λ gradually increased with increasing GOx surface concentrations, the λ value reached 0.34 when it was determined on the surfaces modified by 500 ng/mm2 of GOx. Any statistically significant changes in FB-SECM were detected when the surface concentration of GOx exceeded 50 pg/mm2. Notably, localized electrochemical impedance measurements using the SEIM mode enabled one to detect GOx activity even when GOx was immobilized on a nonconductive substrate surface. The results show that redox competition-based SEIM can be used to determine glucose concentrations in the range of 2-20 mM, while using 10 Hz AC perturbation. An FB-SECM configuration allows the reuse of the ultramicroelectrode while providing the localized impedance-based glucose concentration and enzyme activity measurements.