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

Significance Statement

An electrochemical method for measuring the activity of a cancer-related enzymes such as cathepsin B has been demonstrated by Dr. Jun Li and collaborators at Kansas State University. The method is based on enhanced AC voltammetry using nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). The VACNFs of ~150 nm in diameter and 3 to 5 µm in length were grown on the conductive substrates and embedded in SiO2 matrix. After polishing and plasma etching, controlled number of VACNF tips were exposed to form the VACNF NEA. TetrapeptideH2N-(CH2)4-CO-Leu-Arg-Phe-Gly-NH-CH2-Fc specific to cathepsin B were covalently attached to the exposed VACNF tips. The redox signal of Fc was measured with AC voltammetry (ACV) at a much higher frequency on VACNF NEAs (~ 1 kHz) than the traditional macroscopic glassy carbon electrodes (40 Hz), enabled by VACNF’s unique interior structure.

This method enablde the measurement of the kinetic process of proteolytic cleavage of the surface-attached tetrapeptides by cathepsin B, giving an exponential decay of the ACV peak current versus the reaction time. The exponential component can be analyzed with a heterogeneous Michaelis-Menten model. Quantitative detection of cathepsin B activity has been demonstrated in purified enzyme solution, and complex samples including in the whole lysate of human breast tissue and spiked tissue lysate. The cathepsin B concentration was further validated by spiking it into complex tissue lysates after immunoprecipitation. This method could be developed as a portable multiplex electronic technique for rapid cancer diagnosis, treatment monitoring and inhibitor screening by profiling the activities of a library of proteases in serum or blood samples.

 

 

Journal Reference

Swisher LZ, Prior AM, Shishido S, Nguyen TA, Hua DH, Li J.

Biosens Bioelectron. 2014 Jun 15;56:129-36.

Department of Chemistry, Kansas State University, Manhattan, KS 66506, United States and

Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, United States and

Department of Chemistry, Kansas State University, Manhattan, KS 66506, United States. Electronic address: [email protected]

 

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 (~1kHz) 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) × 10(4)M(-1)s(-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 is ~13.4 nM higher cathepsin Bconcentration 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 × 10(4)M(-1)s(-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.

Copyright © 2014 Elsevier B.V. All rights reserved.

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