Highly sensitive electrochemical detection of DNA hybridisation by coupling the chemical reduction of a redox label to the electrode reaction of a solution phase mediator

Abstract

We have described a highly sensitive method for detecting DNA hybridisation using a redox-labeled stem loop probe. The redox labels were poly(styrene-co-acrylic) (PSA) spheres of 454 nm diameter, modified by methylene blue (MB) deposited alternatively with poly(sodium 4-styrene sulphonate) (PSS) in a layer-by-layer process. Each PSA sphere carried approx. 3.7 × 10⁵ molecules of MB, as determined optically. DIG-tagged stem loop probes were immobilised on screen printed electrodes bearing anti-DIG antibodies. Binding with the target enabled straightening of the stem loop, which made attachment to the MB-coated PSA spheres possible. For measuring the current from the direct reduction of MB by differential pulse voltammetry, a 30 mer DNA target common to 70 strains of Escherichia coli was calibrated across the range 1.0 fM to 100 pM (gradient = 3.2 × 10⁻⁸ A (log fM)⁻¹, r² = 0.95, n = 60), with an LOD of ∼58 fM. By using Fe(CN)₆^3−/4− as a solution phase mediator for the MB reduction, we were able to lower the LOD to ∼39 aM (gradient = 5.95 × 10⁻⁸ A (log aM)⁻¹, r² = 0.96, n = 30), which corresponds to the detection of 0.76 ag (∼50 molecules) in the 2 μL analyte sample. We hypothesise that the lowering of the LOD was due to the fact that not all the MB labels were able to contact the electrode surface.