Hairpin-DNA Probe for Enzyme-Amplified Electrochemical Detection of Legionella pneumophila

Abstract

An electrochemical genosensor for the detection of nucleic acid sequences specific of Legionella pneumophila is reported. An immobilized thiolated hairpin probe is combined with a sandwich-type hybridization assay, using biotin as a tracer in the signaling probe, and streptavidin-alkaline phosphatase as reporter molecule. The activity of the immobilized enzyme was voltammetrically determined by measuring the amount of 1-naphthol generated after 2 min of enzymatic dephosphorylation of 1-naphthyl phosphate. The sensor allows discrimination between L. pneumophila and L. longbeachae with high sensitivity under identical assay conditions (no changes in stringency). A limit of detection of 340 pM L. pneumophila DNA, and a linear relationship between the analytical signal and the logarithm of the target concentration to 2 μM were obtained. Experimental results show the superior sensitivity and selectivity of the hairpin-based assay when compared with analogous sandwich-type assays using linear capture probes.