Electrochemical Coding for Multiplexed Immunoassays of Proteins

An electrochemical immunoassay protocol for the simultaneous measurements of proteins, based on the use of different inorganic nanocrystal tracers is described. The multiprotein electrical detection capability is coupled to the amplification feature of electrochemical stripping transduction (to yield fmol detection limits) and with an efficient magnetic separation (to minimize nonspecific adsorption effects). The multianalyte electrical sandwich immunoassay involves a dual binding event, based on antibodies linked to the nanocrystal tags and magnetic beads. Carbamate linkage is used for conjugating the hydroxyl-terminated nanocrystals with the secondary antibodies. Each biorecognition event yields a distinct voltammetric peak, whose position and size reflects the identity and level, respectively, of the corresponding antigen. The concept is demonstrated for a simultaneous immunoassay of β2-microglobulin, IgG, bovine serum albumin, and C-reactive protein in connection with ZnS, CdS, PbS, and CuS colloidal crystals, respectively. These nanocrystal labels exhibit similar sensitivity. Such electrochemical coding could be readily multiplexed and scaled up in multiwell microtiter plates to allow simultaneous parallel detection of numerous proteins or samples and is expected to open new opportunities for protein diagnostics and biosecurity.