A fluorometric lead(II) assay by using a DNA dendrimer as a carrier for the immobilization of the signal probe.

Abstract

A DNA dendrimer was constructed by combination of rolling circle amplification (RCA) and hybridization chain reaction (HCR). A fluorescence resonance energy transfer (FRET) pair consisting of 6-carboxyfluorescein (FAM) and 5-carboxytetramethylrhodamine (TAMRA) was then created as a signaling probe for the ultrasensitive detection of Pb2+. Firstly, the DNAzyme released by Pb2+-induced recycling amplification as a primer induces RCA to open two different hairpins for generating repeated "Y" structures. Next, two other hairpins (labeled with FAM and TAMRA, respectively) are opened by the "Y" structures to trigger the HCR. As a result, a DNA dendrimer is generated. It is high loading with FRET pair and also promotes FRET pair mutually close to produce a remarkable FRET signal. Hence, ultrasensitive detection of Pb2+ is accomplished by measurement of the ratio of the yellow fluorescence of TAMRA (peakingat 588nm) and the green fluorescence of FAM (at 525nm). The method works in the 0.001 to 10nM Pb2+ concentration range and has a 0.3 pM detection limit. It was applied to determination of intracellular Pb2+ with convincing performance. Graphical abstractSchematic representation of novel DNA dendrimer produced by the combination of rolling circle amplification (RCA) and hybridization chain reaction (HCR) for immobilization of the fluorescence resonance energy transfer (FRET) pair as signal probe forsensitive determination of Pb2+.