Multi-labeled electrochemical sensor for cost-efficient detection of single nucleotide substitutions in folded nucleic acids

Analysis of single nucleotide substitutions (SNS) in nucleic acids is the basis for the diagnosis of drug-resistant pathogens as well as human genetic disorders among a broad range of other applications. However, SNS are often inaccessible for the analysis by hybridization probe due to the location in double stranded regions of folded single stranded DNA or RNA analytes. Here we introduce an electrochemical sensor that is able to analyze SNS in folded nucleic acids. The electrochemical sensor takes advantage of a universal electrode-bound hairpin (UTH). It relies on the recognition of targeted nucleic acids by analyte-specific adaptor strands R and L that also hybridize with UTH. Strand L can bind several methylene blue (MeB)-probes thus placing MeB groups close to the electrode surface, which enables cathodic charge transfer. We demonstrated that the same UTH-functionalized electrode and MeB-probe can be used to analyze DNA analytes with different sequences. The sensor is highly selective toward SNS even at room temperature and can be regenerated for next round by rinse with water. The ability of the sensor to analyze SNS within secondary structure folded DNA was demonstrated. The sensing system is capable of detecting SNS in bacterial DNA, useful to differentiate drug-resistant from drug-susceptible mycobacterium tuberculosis. The proposed platform uniquely combines high SNS selectivity with ability to analyze potentially any DNA or RNA sequence including those folded in stable structures, therefore, creates a basis for a cost efficient electrochemical sensing of nucleic acids applicable both in medical diagnostics and environmental monitoring of microorganisms.