Electrochemical quantification of DNA amplicons via the detection of non-hybridised guanine bases on low-density electrode arrays

Abstract

A new strategy for the electrochemical detection and signal amplification of DNA at gold electrodes is described. Current methodologies for DNA biosensing based on the electrochemical detection of electroactive base-specific labels such as methylene blue (MB) suffer from lengthy incubation and washing steps. Addressing these limitations, we report a novel approach for the electrochemical quantification of surface hybrid, using the control gene LTA, 107 bases long, as a model target. An array of 15 gold electrodes was used to detect the formation of hybridised duplex following interaction of non-hybridised guanine bases with MB present in solution. Upon hybridisation the number of free guanines present at the electrode surface increased from 8 to 25 due to guanine bases present in the target sequence which did not participate in hybridisation and remained free to interact directly with methylene blue. This increase in free guanines consequently concentrated MB directly at the electrode surface. We found that the MB signal recorded for 100 nM of the complementary LTA was typically 2.14 times higher than that of the non-hybridised state. Very low cross-reactivity (<7%) with a non-complementary probe was recorded. The assay was optimised with regards to methylene blue concentration, hybridisation time and regeneration. The assay was quantitative and linear in the range of 6.25-50 nM target DNA exhibiting an LOD of 17.5 nM. The assay was rapid and easy to perform, with no need for lengthy incubations with the methylene blue label or requirement for washing steps. Ongoing work addresses the impact of guanine location on the signal in order to tailor design specific signalling domains of PCR products.