Covalent Hemin/G4 complex-linked sandwich bioassay on magnetic beads for femtomolar HER-2/neu detection in human serum via direct electrocatalytic reduction of oxygen

Abstract

Liquid biopsy assays for tumour biomarkers circulating in blood are perspective non-invasive tools for cancer diagnosis and treatment monitoring. Here, we suggest a simple, 1 h long electrochemical DNAzyme-linked aptamer- and immuno-sandwich magnetic assay for analysis of serum HER-2/neu protein overexpressed in several aggressive cancers. In the assay, we used a covalent hemin-guanine quadruplex (G4) complex as a novel O2-dependent electrocatalytic label that allowed 10 fM (aptamer-aptamer) and 1 fM (aptamer-antibody) detection of HER-2/neu in human serum. The O2 reactivity of the aptamer-conjugated label was detected at high-surface-area graphite electrodes displaying a high efficiency of O2 reduction electro-catalyzed by this DNAzyme. In contrast to the recognised H2O2 reactivity, the O2 reactivity of the covalent hemin/G4 complex depended only on ambient O2 present in solutions, and did not require adding such traditional reagents as hemin and H2O2, and solution de-aeration. Human serum albumin, urokinase plasminogen activator and thrombin did not interfere, and the assay was used for analysis of basal serum levels of HER-2/neu. Due to the simplicity and low cost, sandwich assays exploiting O2-linked electrocatalysis by the covalent hemin-G4 complexes represent a more advanced electrochemical ELISA platform for ultrasensitive and fast detection of low concentrations of proteins in complex biological matrices.