title: Sensitive electrochemical analysis of BRAF V600E mutation based on an amplification-refractory mutation system coupled with multienzyme functionalized Fe3O4/Au nanoparticles.
abstract: 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.
authors: Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR China.
authors: Bo
authors: B
authors: Situ
authors: Nannan
authors: N
authors: Cao
authors: Bo
authors: B
authors: Li
authors: Qinlan
authors: Q
authors: Liu
authors: Li
authors: L
authors: Lin
authors: Zong
authors: Z
authors: Dai
authors: Xiaoyong
authors: X
authors: Zou
authors: Zhen
authors: Z
authors: Cai
authors: Qian
authors: Q
authors: Wang
authors: Xiaohui
authors: X
authors: Yan
authors: Lei
authors: L
authors: Zheng
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques / Q000295:instrumentation*; D003217:Conductometry / Q000295:instrumentation*; D004252:DNA Mutational Analysis / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005290:Ferric Compounds / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D053768:Metal Nanoparticles / Q000737:chemistry*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*
chemical_list: D005290:Ferric Compounds; C000499:ferric oxide; D006046:Gold; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf