Highly sensitive electrochemical detection of genomic DNA based on stem loop probes structured for magnetic collection and measurement via metalised hollow polyelectrolyte shells

We report a highly sensitive method for the electrochemical detection of genomic DNA, based on the employment of two sub-micron oligonucleotide labels - one for magnetic collection and the other for voltammetric detection - and their incorporation onto a stem loop DNA probe. The magnetic label consists of a latex particle of mean diameter 441±6nm, bearing magnetic Fe₃O₄ particles and approx. 3.5×10⁵ anti-DIG antibodies. The voltammetric label is a hollow polyelectrolyte shell containing approx. 1.0×10¹¹ Au atoms in the form of well dispersed Au nanoparticles. A DIG tag on one arm of the stem loop enables binding to the magnetic label, while a thiol tag on the other arm enables attachment to the Au nanoparticles. Due to steric hindrance from the two relatively large labels, attachment of both moieties is dependant on target-probe hybridisation straightening the loop. Once attached, sensitive DNA measurement is facilitated by magnetic collection of the DNA into a small volume and by the high quantity of Au atoms available for detection. Using differential pulse anodic stripping voltammetry we calibrated a 30mer sequence common to 71 strains of Escherichia coli across the concentration range from 0.1aM to 100pM with a LOD of 1.8aM. Three strains of E. coli, BL 21, ATCC8739, O157:H7, when spiked into UHT milk and fermented palm juice, could be detected with LODs of approx. 2-4CFUmL^-1 in an assay time of approx. 140min.