The overexpression of various enzymes such as kinases, phosphatases, and proteases has been recognized to lead to cancers. The enzyme activity profiling can be used for cancer diagnosis, therapeutic monitoring, and drug discovery. Here we propose a label-free electronic method for rapid, ultrasensitive, and highly reliable profiling of the activities of cancerous proteases. We hypothesize that a library of peptides attached to an independently addressed electrode array can be used to profile the activities of a mixture of cancer-related proteases through mapping the electrochemical signals at individual electrodes. An electroactive tag (i.e. a ferrocene, Fc) can be attached to the distal end of each peptide, which produces a characteristic redox signal with amplified amplitude measured by an alternate current (AC) voltammetry technique. This signal is reliable and not as easily interfered by nonspecific factors as other electronic techniques. Upon proteolysis by specific proteases such as legumain and matriptase, the peptides at a specific electrode site will be cleaved and the attached Fc moiety will leave the electrode surface. As a result, the redox signal will decay vs. time with the decay rate inversely proportional to the activity of that particular protease. A novel nanotechnology, i.e. embedded nanoelectrode arrays (NEAs), will be used to enhance the detection speed and sensitivity. The NEAs are based on well- separated vertically aligned carbon nanofibers (VACNFs) grown on individually addressed microelectrode pads and then encapsulated with SiO2. The top surface is polished or plasma etched to expose the very end of the carbon nanofibers (CNFs) of ~100 nm in diameter. Peptide substrates are covalently functionalized to the end of the CNFs. The NEA allows selective functionalization of very small amount of peptide molecules, facilitating extremely sensitive and fast electronic detection using high-frequency (~3 kHz) AC voltammetry techniques. In long term, the NEA can be fabricated into individually addressed multiplex chip, enabling the simultaneous detection of multiple proteases using many (up to 100) peptide substrates using only 5-10 microliter samples. This will significantly expedite protease profiling for cancer diagnosis, staging, outcome prediction, prognosis, and treatment selection.
Three specific aims are designed within the scope of this AREA application toward our long-term goals:
Specific Aim 1 : CNF NEA fabrication and property characterization, Specific Aim 2: Method development for detecting peptide proteolysis with legumain and matriptase, Specific Aim 3: Protease profiling of various breast cancer cell lines. This AREA application will facilitate the transition of nanobiosensors research to potential clinical cancer diagnosis and therapeutic monitoring by rapid profiling cancer-related proteases through the collaboration of three researchers in physical sciences and cancer biology. It will stimulate graduate and undergraduate students at Kansas State University for further career development in health sciences and related fields through research experience in this cancer detection project.

Public Health Relevance

Reliable, highly specific, ultrasensitive, and low-cost electronic technology is important for rapid screening of cancer diseases and monitoring cancer treatments. This proposed work is to develop a nanotechnology based electronic method for cancer detection through rapid profiling of the activities of cancerous proteases. This will significantly expedite cancer diagnosis, staging, outcome prediction, prognosis, and treatment selection.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Academic Research Enhancement Awards (AREA) (R15)
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Nanotechnology Study Section (NANO)
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Knowlton, John R
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Kansas State University
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