Quantitative electrochemical detection of cathepsin B activity in complex tissue lysates using enhanced AC voltammetry at carbon nanofiber nanoelectrode arrays

Abstract

The proteolytic activity of a cancer-related enzyme cathepsin B is measured with alternating current voltammetry (ACV) using ferrocene (Fc) labeled tetrapeptides attached to nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). This combination enables the use of high AC frequencies (~1 kHz) with enhanced electrochemical signals. The specific proteolysis of the Fc-peptide by cathepsin B produces decay in the ACV peak current versus the reaction time. The exponential component of the raw data can be extracted and defined as the “extracted proteolytic signal” which allows consistent quantitative analyses using a heterogeneous Michaelis-Menten model. A “specificity constant” kcat/KM = (3.68 ± 0.50) × 104 M−1s−1 for purified cathepsin B was obtained. The detections of cathepsin B activity in different concentrations of whole lysate of human breast tissue, tissue lysate spiked with varied concentrations of cathepsin B, and the tissue lysate after immunoprecipitation showed that there are ~13.4 nM higher cathepsin B concentration in 29.1 μg·mL−1 of whole tissue lysate than the immunoprecipitated sample. The well-defined regular VACNF NEAs by e-beam lithography show a much faster kinetics for cathepsin B proteolysis with kcat/KM = 9.2 × 104 M−1s−1. These results illustrate the potential of this technique as a portable multiplex electronic system for cancer diagnosis by rapid protease profiling of serum or blood samples.

Quantitative electrochemical detection of cathepsin B activity in breast cancer cell lysates using carbon nanofiber nanoelectrode arrays toward identification of cancer formation

“…After adding cathepsin B, the tetrapeptide was cleaved at the site between arginine and phenylalanine, which has been proved by HPLC and MS analyses in our previous study. 14, 15 The fragment containing Fc was thus released into the bulk solution causing the peak current i p,acv to decrease (Figure S2B). The kinetics of the proteolysis can be monitored by continuously repeated ACV measurements (Figure S2C).…”

“…15, 16, 22 According to our previous study, the variation can be normalized by using the relative change represented by the “extracted proteolytic signal” S, as defined below: 15 …”

“…The proteolysis kinetics on the VACNF NEAs can be explained with a modified Michaelis-Menten model for heterogeneous enzymatic reactions 14, 15, 23 :…”

“…14, 15, 18-21 Briefly, randomly distributed VACNFs with average length of ∼5 μm and an areal density of ∼1×10 9 CNFs/cm 2 were grown on Cr coated Si(100) wafer using a plasma enhanced chemical vapor deposition (PECVD) system (Aixtron, CA, USA). A nickel film with ∼22 nm thickness was coated as the catalyst for CNF growth.…”

“…In previous studies, we have demonstrated an electrochemical technique in detecting the proteolytic kinetics of two cancer-related proteases legumain and cathepsin B in purified solutions and complex whole tissue lysates, respectively, using nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs), a special type of carbon nanotubes. 14, 15 Specific tetrapeptides with a ferrocene (Fc) group at the distal end were attached to the exposed VACNF tip and selectively cleaved by the proteases. The unique structure of VACNFs allowed using a high-frequency (∼1 kHz) alternating current voltammetry (ACV) to monitor the small electrochemical signal from Fc.…”