期刊
JOURNAL OF PHYSICAL CHEMISTRY C
卷 117, 期 8, 页码 4268-4277出版社
AMER CHEMICAL SOC
DOI: 10.1021/jp312031u
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资金
- National Cancer Institute [R15CA159250]
- National Center for Research Resources [P20RR015563]
- Johnson Center for Basic Cancer Research at Kansas State University
- NATIONAL CANCER INSTITUTE [R15CA159250] Funding Source: NIH RePORTER
- NATIONAL CENTER FOR RESEARCH RESOURCES [P20RR015563] Funding Source: NIH RePORTER
We report an electrochemical method for measuring the activity of proteases using nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). The VACNFs of similar to 150 nm in diameter and 3-5 pm in length were grown on conductive substrates and encapsulated in SiO2 matrix. After polishing and plasma etching, controlled VACNF tips are exposed to form an embedded VACNF NEA. Two types of tetrapeptides specific to cancer-mediated proteases legumain and cathepsin B are covalently attached to the exposed VACNF tip, with a ferrocene (Fc) moiety linked at the distal end. The redox signal of Fc can be measured with AC voltammetry (ACV) at similar to 1 kHz frequency on VACNF NEAs, showing distinct properties from macroscopic glassy carbon electrodes due to VACNF's unique interior structure. The enhanced ACV properties enable the kinetic measurements of proteolytic cleavage of the surface-attached tetrapeptides by proteases, further validated with a fluorescence assay. The data can be analyzed with a heterogeneous Michaelis Menten model, giving specificity constant k(cat)/K-m as (4.3 +/- 0.8) X 10(4)M(-1)s(-1) for cathepsin B and (1.13 +/- 0.38) X 10(4) M-1 s(-1) for legumain. This method could be developed as portable multiplex electronic techniques for rapid cancer diagnosis and treatment monitoring.
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