Proximity Hybridization-Regulated Immunoassay for Cell Surface Protein and Protein-Overexpressing Cancer Cells via Electrochemiluminescence.

A simple electrochemiluminescence (ECL) immunoassay based on a proximity hybridization-regulated strategy was developed for highly sensitive and specific detection of cell surface protein and protein-overexpressing cancer cells. A biosensor was fabricated by self-assembling a thiolated capture ss-DNA3 (partially hybridize with ss-DNA1 and ss-DNA2) and blocking with 6-mercapto-1-hexanol on a gold electrode surface. Target protein was simultaneously bound by two ss-DNA-tagged antibody probes (DNA1-Ab1 and DNA2-Ab2), while DNA1 and DNA2 were brought in sufficient proximity and hybridized with capture DNA3 on the surface of the biosensor. After ECL signal reagent Ru(phen) was intercalated into the hybridized ds-DNAs, ECL measurement was performed in the coreactant solution. A "signal on" proximity hybridization-regulated ECL immunoassay for alpha-fetoprotein (AFP) was developed. The ECL intensity increased with the increase of AFP concentration in the range of 0.05-20.0 ng/mL with a detection limit of 6.2 pg/mL. Moreover, the developed ECL method was successfully used to detect AFP-overexpressing cancer cells (MCF-7 cancer cells as model) with a detection limit of 620 cells/mL (∼60 MCF-7 cells in 100 μL of cell suspension) and discriminate AFP expression on different types of the living cell surface. This work for the first time reports a proximity hybridization-regulated ECL immunoassay for the detection of the cell surface protein on a living cell surface with good specificity and sensitivity. This simple, specific, and sensitive strategy is greatly promising for the detection of proteins and specific cells.