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.

A comprehensive review summarizing the recent biomedical applications of functionalized carbon nanofibers

“…Nanoelectrode arrays (NEAs) designed with vertically aligned CNFs (VCNFs) act as an excellent electrochemical platform to detect protease activity, as presented by Swisher et al 48 . The same research group developed a protruding VCNFs NEAs contained peptides substrate and tagged with ferrocene label (FC) 49 . Among different peptides with different lengths, hexapeptide H 2 N(CH 2 ) 4 CO‐Pro‐Leu‐Arg‐Phe‐Gly‐Ala‐NHCH 2 Fc was the ideal probe to detect and measure Cathepsin B.…”

Micro/Nano Electrode Array Sensors: Advances in Fabrication and Emerging Applications in Bioanalysis

“…In addition, nucleation and growth of materials to form nanostructures with electrochemical performances has also become a way for construction of nano electrode arrays. Vertically aligned carbon nanotubes/nanofibers (Arumugam et al, 2009;Robinson et al, 2016;Song et al, 2019) as well as a vast range of nano electrocatalysts (e.g., prussian blue, porous gold or platinum nanowire, NiO nanocone, mesoporous rhombus-shaped ZnO rod, etc.) have been fabricated as nano electrode arrays (Karyakin et al, 2004;Puganova and Karyakin, 2005;Zhang et al, 2009;Wang et al, 2011Wang et al, , 2012Wen et al, 2015).…”

“…can be detected by micro/nano electrode array sensors (Koehne et al, 2004;Lapierre-Devlin et al, 2005;Periyakaruppan et al, 2013;Silvestrini et al, 2013;Selvam et al, 2015;Delle et al, 2018;Song et al, 2019). Song et al (2019) developed vertically aligned carbon nanofibers as unique electrochemical platform for investigating protease activities (Figure 2A). The carbon nanofibers are functionalized with specific peptide substrates containing a ferrocene tag.…”

“…Owing to the breakthrough in new materials, microelectronic technology as well as electrochemical understandings, biosensors based on micro/nano electrode arrays are moving toward miniaturization, digitization, intelligence and systematization. Smart sensing created from miniaturization of portable micro/nano electrode array sensors as well as wearable intelligent devices has been paid great attention in recent years (Triroj FIGURE 2 | (A) Carbon nanofiber nanoelectrode arrays and their electrochemical activity for protease analysis (Song et al, 2019). Copyright © 2019 American Chemical Society.…”

Antibody-Electroactive Probe Conjugates Based Electrochemical Immunosensors

“…The latest progress in the area of electrochemical immunosensors has been related to the modification of working electrode surfaces using novel nanomaterials in order to obtain superior sensor sensitivity [23]. Significantly low detection limits could be obtained using nanoelectrode ensembles (NEEs) [24][25][26][27]. These electrodes are characterized by a detection limit of 2-3 orders magnitude lower than a macroelectrode with the same geometric area.…”