Carbon nanotube microwell array for sensitive electrochemiluminescent detection of cancer biomarker proteins

This paper describes fabrication of a novel electrochemiluminescence (ECL) immunosensor array featuring capture-antibody-decorated single-wall carbon nanotube (SWCNT) forests residing in the bottoms of 10-microL wells with hydrophobic polymer walls. Silica nanoparticles containing [Ru(bpy)(3)](2+) and secondary antibodies (RuBPY-silica-Ab(2)) are employed in this system for highly sensitive two-analyte detection. Antibodies to prostate specific antigen (PSA) and interleukin-6 (IL-6) were attached to the same RuBPY-silica-Ab(2) particle. The array was fabricated by forming the wells on a conductive pyrolytic graphite chip (1 in. × 1 in.) with a single connection to a potentiostat to achieve ECL. The sandwich immunoassay protocol employs antibodies attached to SWCNTs in the wells to capture analyte proteins. Then RuBPY-silica-Ab(2) is added to bind to the captured proteins. ECL is initiated in the microwells by electrochemical oxidation of tripropyl amine (TprA), which generates excited state [Ru(bpy)(3)](2+) in the 100-nm particles, and is measured with a charge-coupled device (CCD) camera. Separation of the analytical spots by the hydrophobic wall barriers enabled simultaneous immunoassays for two proteins in a single sample without cross-contamination. The detection limit (DL) for PSA was 1 pg mL(-1) and for IL-6 was 0.25 pg mL(-1) (IL-6) in serum. Array determinations of PSA and IL-6 in patient serum were well-correlated with single-protein ELISAs. These microwell SWCNT immunoarrays provide a simple, sensitive approach to the detection of two or more proteins.