Nanomaterial-Based Bioelectronic Detection of Disease Markers (2009 - Present)

Investigator:  Bernard Munge, Salve Regina University
Mentor:  James Rusling, University of Connecticut

Abstract:  This Proposal addresses the Area of Biomedical Diagnostics for early Detection of Cancer. Specific detection of cancer biomarker proteins in serum is critically important for early cancer detection, leading to greatly improved patient prognoses, treatment success, and even cancer prevention. High accuracy is vital in simultaneous detection of collection biomarker proteins for specific forms of cancer. Our goal is to develop nanomaterial-based arrays to measure collections of early cancer biomarker proteins for specific forms of cancer. Such devices feature vertical "forests" of single-walled carbon nanotubes (SWNT) with capture antibodies attached. The nanotube forests will be fabricated into immunosensor arrays featuring electrochemical detection of biomarker proteins via enzyme labels on secondary anti-bodies.  This project combines efficient electrical transduction of single walled carbon nanotubes with ~1.4 nm diameters and the world’s highest conductivity per unit mass with biomolecular recognition by antibodies. To date, there are a number of established protocols for assembling 20-100 nm vertically aligned carbon nanotubes arrays on surfaces in nm-scale bundles called SWNT forests acting as electrical conduits between conductive surface and active bio-molecules. SWNT forests provide high surface area for attachment of antibodies, leading to highly sensitive detection. Electronic devices offer elegant ways for interfacing biorecognition and transduction events and are uniquely qualified for meeting the high sensitivity and portability requirements of future biodetection systems. Electrochemical bioassays involving enzyme tracers have received particular attention due to the intrinsic signal amplification provided by bio-catalytic reactions. Recently, we used a sandwich immunological assay with antibody-antigen captured on magnetic beads and LBL enzyme tracers loaded on CNT, maximizing the number of enzyme tracers per binding event to achieve signal amplification. Such amplified bioelectronic assays allowed detection of DNA and proteins down to 80 copies (5.4 aM) and 2000 protein molecules (67 aM), respectively. In our preliminary results we demonstrated prototype immunosensor based on SWNT forest with excellent detection limits of 0.25 Fmol mL-1 for prostate specific cancer biomarker in serum using giant molecular tag, CNT-HRP-Ab2 conjugates with HRP/Ab2 ~ 300 for signal amplification. The peroxidase-linked amperometric immunoassay used enzyme catalyzed reduction of H2O2 to provide a large signal transmitted through SWNT.  This prototype sensor is better than all competing immunoassays for PSA, and offer great promise for ultra-sensitive detection of disease biomarkers. Such a sensor protocol require tiny sample and is a rapid simple process amenable to immunoarray fabrication. To achieve highest sensitivity, we will further eliminate non-specific binding (NSB), optimizing the signal amplification protocol by exploring competitively adsorbed protein/detergent additives. Combining ultra-low NSB with electrical wiring of nanotubes and multiple enzyme labels strategies promise low detection limits and high sensitivity. Finally, we will integrate optimized fabrication/detection approaches into multi-protein SWNT-antibody arrays for collections of cancer biomarkers.

Presentations

2008 - 2009

Munge, B.S., Fisher, F., Millord, L., Krause, C. 2008. Nanomaterials-based immunoassay for sensitive electrochemical detection of matrix metalloproteinase-3 (MMP-3), cancer biomarker in serum. 2nd Biennial National IDeA Symposium of Biomedical Research Excellence, Washington D.C., August 6-8.

Munge, B.S., Krause, C., Dowd, R. 2008. Sensitive electrochemical immunoassay of a biomarker alpha-fetoprotein based on carbon nanotubes. 2nd Biennial National IDeA Symposium of Biomedical Research Excellence, Washington D.C., August 6-8.

Maricic, P., Hall, A., Munge, B.S. 2008. Integrated carbon nanotube-polyguanine functionalized polymeric nanoparticles for sensitive immunodetection of prostate specific antigen (PSA) in serum. 2nd Biennial National IDeA Symposium of Biomedical Research Excellence, Washington D.C., August 6-8 

Munge, B.S., Fisher, J., Millord, L., Krause, C. 2008. Polymeric beads amplification strategy for sensitive electrochemical detection of matrix metalloproteinase-3, a cancer biomarker protein. Rhode Island Research Alliance Symposium, Providence, RI, June 3.

2007 - 2008

Munge, B., Fisher, J. 2007. Sensitive immunoassay of matrix metalloproteinase-2, a cancer biomarker protein based on vertically-aligned single-wall carbon nanotubes arrays. 2nd Northeast Regional IDeA Meeting, Burlington, VT, August 15-17.

Leonard, K., Munge, B. S. 2007. Electrochemical detection of prostate carcinoma biomarkers using nanotechnology. COBRE/INBRE Symposium. Providence, RI, May 30.