Methylene blue as an electrochemical indicator for DF508 cystic fibrosis mutation detection

Abstract

Cystic fibrosis is one of the most common life-shortening, childhood-onset inherited diseases. Among the 1,000 known cystic fibrosis-related mutations, DF508 is the most common, with a frequency varying between 50% and 70% according to geographical areas and population typology. In this work, we report the use of methylene blue as an electrochemical reporting agent in the discrimination of synthetic PCR analogue of the DF508 cystic fibrosis mutation (Mut) from the wild type (Wt). At optimum experimental condition, a discrimination factor between mutant and wild type of approximately 1.5-fold was found. The proposed assay was quantitative and linear in the range of 10-100 nM, exhibiting a limit of detection of 2.64 nM. Electrochemical studies at variable ionic strength conditions allowed further elucidation of the mechanism of the methylene blue (MB)-DNA interaction. To the best of our knowledge, this is the first report of detection of hybridisation solely via guanine-specific MB-DNA interaction simultaneously in MB solution, independent of electrostatic interaction as demonstrated in the ionic strength study. The introduction of formamide in the hybridization buffer, to improve discrimination, was also investigated. Finally, mutant wild type discrimination was demonstrated, at 10 nM concentration, with the use of a multi-sensor setup.

Electrochemical detection of foodborne pathogen Aeromonas hydrophila by DNA hybridization biosensor

“…Methylene blue (MB), an organic dye classified into the phenolthiazine family, is an example of such indicator (Yang et al, 2002). It was used as a reporting element in several electrochemical genosensors already presented in the literature (Tichoniuk et al, 2008;Nasef et al, 2010).…”

Novel DNA Biosensor for Direct Determination of Carrageenan

“…In general, there are three different basic interaction mechanisms between MB and DNA, which have been identified to be (1) electrostatic interaction of cationic charge in MB with negatively charged phosphate backbone DNA, (2) MB intercalation within dsDNA double-helix (3) and MB binding with the exposed guanine bases on ssDNA 31 . Among the three ways of MB-DNA interactions, intercalation of MB into dsDNA gives the highest redox peak current signal 32 .…”

Application of titanium dioxide nanowires and electroreduced graphene oxide modified electrodes for the electrochemical detection of specific tlh gene sequence from Vibrio parahaemolyticus

“…MB can also bind specically to the guanine bases on the ssDNA sequence. [41][42][43][44] Aer the hybridization reaction with the formation of dsDNA structure on the electrode surface, the electrochemical response of MB is decreased due to the inaccessibility of MB to the guanine bases of dsDNA. Electrochemical behavior of MB on different DNA modied electrodes were investigated with the results shown in Fig.…”