RGB colour coding of Y-shaped DNA for simultaneous tri-analyte solid phase hybridization detection

We present a new concept for tri-analyte DNA detection based on the idea of a Y-shaped capture probe which, after tri-target and fluorescently labeled reporter probe binding, becomes colour-coded to generate images in an RGB colour scheme. Hence, the RGB value of the resulting secondary pseudo-colour presented by the hybridized Y-DNA can be related to the ratio of the primary pseudo-colours present in its make-up, and thus to the ratio of the three target concentrations. As a proof of concept we detect sequences from the genes of the pathogenic bacterial strains Escherichia coli O157:H7, Vibrio cholera and Salmonella enteric in a semi-quantitative manner across the range 20–167 nM. The assay was relatively quick, with a time from hybridization to completed data interpretation of approximately 4 h.