September 9, 2011
Journal Article

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

Abstract

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 multi-enzymes amplification strategy using gold nanorods (AuNRs) as nanocarrier for co-immobilization of horseradish peroxidase (HRP) and detection antibody (Ab2) at high ratio of HRP/Ab2, 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. The disposable immunosensor array shows excellent promise for clinical screening of phosphorylated proteins and convenient point-of-care diagnostics.

Revised: September 16, 2011 | Published: September 9, 2011

Citation

Du D., J. Wang, D. Lu, A. Dohnalkova, and Y. Lin. 2011. Multiplexed Electrochemical Immunoassay of Phosphorylated Proteins Based on Enzyme-Functionalized Gold Nanorod Labels and Electric Field-Driven Acceleration. Analytical Chemistry 83, no. 17:6580-6585. PNNL-SA-81735. doi:10.1021/ac2009977