Sensitive electrochemical analysis of BRAF V600E mutation based on an amplification-refractory mutation system coupled with multienzyme functionalized Fe3O4/Au nanoparticles

A novel electrochemical biosensor was developed for the analysis of BRAF V600E mutation in colorectal cancer cell samples based on a dual amplification strategy of amplification-refractory mutation system (ARMS) PCR and multiple enzyme labels. The labeled amplicons were conjugated on Fe3O4/Au nanoparticles using Au–S linkages. Alkaline phosphatases were then loaded onto the nanoparticles through biotin–streptavidin interactions. The resultant composite nanoparticles were characterized by transmission electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. In the presence of 2-phospho-l-ascorbic acid, the mutant alleles were quantified on a screen-printed carbon electrode (SPCE) from the anodic current of the enzymatic product, ascorbic acid. BRAF V600E mutant alleles concentrations as low as 0.8% were successfully determined in an excess of wild-type background. In a cell-line dilution model, the proposed method was more sensitive than were DNA sequencing and agarose gel electrophoresis. This work demonstrates a new strategy for sensitivly detecting BRAF V600E variations. It can pave the way for analyzing other rare mutations in complex cancer samples because of its high sensitivity, simplicity, low cost, and easy validation of assay procedures.

► A novel electrochemical biosensor for BRAF V600E mutation in colorectal cancer cell was established.
► A dual amplification strategy of ARMS PCR and multienzyme functionalized Fe3O4/Au nanoparticles was developed and evaluated.
► As low as 0.8% of BRAF V600E mutations were detectable among an excess of wild-type background.
► This strategy can easily be adapted to discern other hotspot mutations in clinical specimens.