Herein we report on an ultrasensitive immunosensor based on glutathione protected gold nanoparticle (GSH-AuNP) for the electrochemical detection of interleukin 8 (IL-8), cancer biomarker in calf serum and proof of concept IL-8 detection in HNSCC cells. GSH-AuNP were bioconjugated to the primary antibodies (Ab1) and used to capture human IL-8 in a sandwich electrochemical immunoassay coupled to horseradish peroxidase enzyme labels. Using the optimized concentrations of the primary (Ab1) and secondary antibodies (Ab2), two sensor approaches were used to measure ultra low (≤ 500 fg mL-1) and elevated levels of IL-8. Biotinylated Ab2 bound to streptavidin HRP with 14-16 labels per antigen was used to measure high IL-8 concentration with a DL of 10 pg mL-1 (1.0 pM) in 10 L calf serum. The second approach greatly amplified the signal using 1 m magnetic beads coated with over 500,000 HRP labels providing the highest sensitivity of (1061.8 nA mL (fg IL-8)-1 cm-2 and the best detection limit of 1 fg mL-1 (100 aM) for IL-8 in 10 L calf serum. This represents a 10,000-fold and 30,000-fold decrease in the DL over the Ab-HRP(14-16) system and the industry standard ELISA for IL-8 respectively. The immuonsensors were also used to accurately measure IL-8 in HNSCC cell lines with excellent correlation to the standard enzyme linked immunosorbent assay (ELISA). These GSH-AuNP based immuonsensors show great promise for the fabrication of ultrasensitive biosensor microarrays for point-of-care cancer diagnosis
