Multiplexed Electrochemical Immunoassay of Phosphorylated Proteins Based on Enzyme-Functionalized Gold Nanorod Labels and Electric Field-Driven Acceleration

A multiplexed electrochemical immunoassay integrating enzyme amplification and electric field-driven strategy was developed for fast and sensitive quantification of phosphorylated p53 at Ser392 (phospho-p53(392)), Ser15 (phospho-p53(15)), Ser46 (phospho-p53(46)), and total p53 simultaneously. The disposable sensor array has four spatially separated working electrodes, and each of them is modified with different capture antibody, which enables simultaneous immunoassay to be conducted without cross-talk between adjacent electrodes. The enhanced sensitivity was achieved by a multienzyme amplification strategy using gold nanorods (AuNRs) as nanocarrier for coimmobilization of horseradish peroxidase (HRP) and detection antibody (Ab(2)) at a high ratio of HRP/Ab(2), which produced an amplified electrocatalytic response by the reduction of HRP oxidized thionine in the presence of hydrogen peroxide. The immunoreaction processes were accelerated by applying +0.4 V for 3 min and then -0.2 V for 1.5 min; thus, the whole sandwich immunoreactions could be completed in less than 5 min. Under optimal conditions, this method could simultaneously detect phospho-p53(392), phospho-p53(15), phospho-p53(46), and total p53 ranging from 0.01 to 20 nM, 0.05 to 20 nM, 0.1 to 50 nM, and 0.05 to 20 nM with detection limits of 5 pM, 20 pM, 30 pM, and 10 pM, respectively. Accurate determinations of these proteins in human plasma samples were demonstrated by comparison to the standard ELISA method. The disposable immunosensor array shows excellent promise for clinical screening of phosphorylated proteins and convenient point-of-care diagnostics.