Electrochemical Activity Assay for Protease AnalysisUsing Carbon Nanofiber Nanoelectrode Arrays

There is a strong demand for bioanalytical techniques to rapidly detect protease activities with high sensitivity and high specificity. This study reports an activity-based electrochemical method toward this goal. Nanoelectrode arrays (NEAs) fabricated with embedded vertically aligned carbon nanofibers (VACNFs) are functionalized with specific peptide substrates containing a ferrocene (Fc) tag. The kinetic proteolysis curves are measured with continuously repeated ac voltammetry, from which the catalytic activity is derived as the inverse of the exponential decay time constant based on a heterogeneous Michaelis–Menten model. Comparison of three peptide substrates with different lengths reveals that the hexapeptide H2N–(CH2)4–CO–Pro-Leu-Arg-Phe-Gly-Ala–NH–CH2–Fc is the optimal probe for cathepsin B. The activity strongly depends on temperature and is the highest around the body temperature. With the optimized peptide substrate and measuring conditions, the limit of detection of cathepsin B activity and con...