Sensitive detection of proteins and bacteria is of central importance in biomedical research, with applications to elucidating disease pathways, immunology, medical diagnostics, systems biology, functional proteomics, and new drug development. Highly selective and sensitive binding-assay arrays are increasingly important in light of emerging areas of biomedical science and intensified world bioterrorism. Sensitive, rapid, specific, multiple detection of proteins, bacteria and toxins in biological and environmental samples is critically important for public health. The broad, long term goals of this proposal are to develop highly sensitive micro- and nano-biosensor arrays for proteins and bacteria using patterned single-walled carbon nanotubes (SWNTs) and biomolecule recognition elements. Major specific applications include simultaneous measurement of suites of protein biomarkers in serum for early cancer detection. Single walled carbon nanotubes (SWNTs) with approximately 1.4 nm diameters have the world's highest conductivity per unit mass and have excellent potential for electrical sensing of biomolecules. We have assembled approximately 30 nm long carbon nanotubes standing in 20-200 nm diameter bundles called SWNT forests with electrical links to conductive surfaces. These high surface area, patternable, conductive nanostructures provide new opportunities for highly sensitive biosensor arrays. In this project, we combine nanotube electrical transduction with specific molecular recognition of antibodies for proteins and bacteria. SWNTs as molecular wires will shuttle electrons vectorially between enzyme labels at their tips and external electrical connections. We focus here on peroxidase-linked amperometric immunoassays, in which enzyme catalyzed reduction of H2O2 provides electrical signals transmitted via SWNTs. In preliminary work, we attached antibodies to SWNT forests and achieved detection of several antigens in the ng/mL range and below. Using newly developed conjugates of nanotubes, peroxidases and secondary antibodies (Ab2) with high label-to-Ab2 ratios with the SWNT immunosensors, we recently detected prostate specific antigen (PSA) in human serum at 0.01 ng/mL (0.25 Fmol/mL), a detection limit better than the best commercial assays for PSA. Ultra-low NSB combined with conductive polymer wiring of nanotube junctions, along with multi-enzyme label strategies, promise the highest possible sensitivity. Thus, we will optimize the new Ab2 nanoconjugates, explore conductive polymer wiring strategies, and eliminate non-specific binding (NSB) of interferences via competitively adsorbed protein/surfactant additives and synthesis of novel low NSB organic surfaces on nanotube ends. Finally, we will integrate optimized fabrication/detection approaches into multi-analyte sensor arrays utilizing patterned SWNT-antibody assemblies. In addition to achieving our specific aims, this project should provide fundamental guidance for fabrication of sensitive microarray devices for a wide variety of biomedical applications.
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