Single-nucleotide polymorphisms (SNPs), insertion/deletion (indel) polymorphisms, and DNA methylation are the most frequent types of genetic variations. As such, DNA polymorphisms play significant roles in genetic mapping and diagnostics. Thus, analytical methods enabling DNA polymorphism detection will provide an invaluable means for early disease diagnosis. However, no single electrochemical nucleic acid-based sensor has achieved the detection of the three major polymorphisms (SNPs, indel polymorphisms, and DNA methylation) with sufficient specificity and sensitivity. In response, we explore the utilization of a catalytic reaction between methylene blue (MB) covalently linked to surface-bound nucleic acid and freely diffusing ferricyanide (Fe(CN)63-) to improve specificity and sensitivity of DNA polymorphism detection. We find that the dynamics of the nucleic acid tether is an additional rate-limiting factor for the electrocatalytic reaction, in addition to the more traditional kinetic and excess factors. Our proof-of-concept experiments demonstrate that the use of electrocatalysis enables differentiation of the three polymorphisms when target sequences are present at 10 nM. We hypothesize that this ability is a result of the distinct dynamics of the DNA probe with each respective polymorphism. In addition to the specificity the sensor displays, the sensor achieves a 20 pM limit of detection. We believe that the electrocatalysis between nucleic acid-tethered MB and Fe(CN)63- is highly promising for electrochemical nucleic acid-based sensors to achieve better specificity and sensitivity.
Keywords: DNA polymorphisms; electrocatalysis; electrochemical nucleic acid-based sensors; ferricyanide; methylene blue.