Nanostructured mesoporous gold electrodes detect protein phosphorylation in cancer with electrochemical signal amplification

Protein phosphorylation is a posttranslational modification of kinase proteins that changes protein’s conformation to regulate crucial biological functions. However, phosphorylation of protein is significantly altered during cancer progression which triggers abnormal cellular pathways and can serve as an emergent diagnostic and prognostic biomarker for cancer. Herein, we develop a nanostructured mesoporous gold electrode (NMGE) that enables highly sensitive detection of protein phosphorylation with electrochemical signal amplification. The biosensor comprises nanostructured mesoporous gold electrodes with a superior electroconductive framework than the nonporous electrodes. We characterize our developed nano/mesoporous gold electrode with various electrochemical methods in the presence of [Fe(CN)6]3-/4 redox system. We find that the mesoporous gold electrode catalyzes both the oxidation and reduction process of [Fe(CN)6]3-/4 system and generates 3-times higher current signal than the non-porous gold electrode. The superior signal transduction of our nano/mesoporous gold electrode is enabled through pore-induced i) high electrochemically active surface area, and ii) reduced impedance with high signal to noise ratio. The assay utilizes direct adsorption of immunoprecipitated purified BRAF protein towards the mesoporous gold electrode and thus avoids cumbersome sensor surface functionalization. Our developed biosensor detects phosphorylated BRAF protein with 2.5-folds increase in sensitivity and ≈10-folds increase in the limit of detection (LOD) in comparison to the nonporous gold electrodes. The assay also works on a wide dynamic range from 0.5-20 ng/µl of protein which further shows its ability for clinical application. We envisage that this nanostructured mesoporous gold biosensor will be of high interest for clinical application.