Hairpin-DNA probe for enzyme-amplified electrochemical detection of Legionella pneumophila.
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 muM 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.
Miranda-Castro
Rebeca
R
Departamento de Química Física y Analítica, Universidad de Oviedo, C/JuliAn Clavería, 8. 33006. Oviedo, Principado de Asturias, Spain.
de-Los-Santos-Alvarez
Patricia
P
Lobo-Castañón
M Jesús
MJ
Miranda-Ordieres
Arturo J
AJ
Tuñón-Blanco
Paulino
P
D001483:Base Sequence; D001710:Biotin / Q000737:chemistry; D015342:DNA Probes / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D004563:Electrochemistry; D005784:Gene Amplification / Q000235:genetics*; D016952:Legionella pneumophila / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation
D015342:DNA Probes; D001710:Biotin