Electrochemical Immunoassay with Microscopic Immunomagnetic Bead Domains and Scanning Electrochemical Microscopy

The formation of mound-like microscopic domains of biochemically active paramagnetic beads reported earlier is extended towards a miniaturized sandwich enzyme immunoassay for the model analyte mouse-IgG. After mouse-IgG is captured by the primary anti-mouse-IgG antibody (Ab) coated magnetic beads of 2.8 µm in diameter, a second anti-mouse-IgG Ab conjugated to an enzyme-label is bound to the analyte. The enzyme label alkaline phosphatase (ALP) is used to catalyze the hydrolysis of 4-aminophenyl phosphate (PAPP) to 4-aminophenol (PAP). The enzymatic activity, which depends on the concentration of captured mouse-IgG, is recorded by scanning electrochemical microscopy (SECM) where a 10 µm Pt electrode scanned over the bead domains detects PAP by oxidation. The anodic current correlates with the concentration of mouse-IgG.