Electrochemical Activity Assay for Protease Analysis Using Carbon Nanofiber Nanoelectrode Arrays.

Abstract

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 concentration can reach 2.49 × 10-4 s-1 and 0.32 nM, respectively. The peptide substrates show high specificity to the cognate proteases, with negligible cross-reactions among three cancer-related proteases cathepsin B, ADAM10, and ADAM17. This electrochemical method can be developed into multiplex chips for rapid profiling of protease activities in cancer diagnosis and treatment monitoring.

• Authors: Yang Song, Huafang Fan, Morgan J Anderson, Jestin Gage Wright, Duy H Hua, Jessica Koehne, M Meyyappan, Jun Li
• Journal: Analytical chemistry (Anal Chem) Vol. 91 Issue 6 Pg. 3971-3979 (03 19 2019) ISSN: 1520-6882 [Electronic] United States
• PMID: 30726059 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• Membrane Proteins
• Carbon
• Amyloid Precursor Protein Secretases
• CTSB protein, human
• Cathepsin B
• ADAM10 Protein
• ADAM10 protein, human
• ADAM17 Protein
• ADAM17 protein, human

Topics

• ADAM10 Protein (analysis, metabolism)
• ADAM17 Protein (analysis, metabolism)
• Amyloid Precursor Protein Secretases (analysis, metabolism)
• Carbon (chemistry)
• Cathepsin B (analysis, metabolism)
• Electrochemical Techniques (methods)
• Electrodes
• Humans
• Membrane Proteins (analysis, metabolism)
• Nanofibers (chemistry)
• Nanotechnology
• Proteolysis