A Simple and Fast Assay Based on Carboxyfluorescein-Loaded Liposome for Quantitative DNA Detection

The development of an innovative and easy way torun assays for the quantitative detection of DNA present inbiologicalfluids (i.e., blood, urine, and saliva) is of great interest forearly diagnosis (e.g., tumors) and personalized medicine. Herein, anew quantitative assay based on the use of highly sensitivecarboxyfluorescein-loaded liposomes as signal amplification systemsis reported. The method has been tested for the detection of lowamounts of DNA sequences. The reported proof of conceptexploits a target DNA molecule as a linker between twocomplementary oligonucleotides. One oligonucleotide is biotinylated at its 3′end and binds to streptavidin-coupled magneticbeads, whereas the other one is conjugated to a cholesterol molecule incorporated in the phospholipidic bilayer of thefluorescent liposomes. In the presence of the target fragment, the correct formation of a construct takes place as witnessed by astrongfluorescence signal, amplified by dissolving lipidic nanoparticles with Triton X-100. The system is able to detect specificnucleotide sequences with a very low detection threshold of target DNA (tens of picomolar). The assay allows the detection ofboth single- and double-stranded DNA. Studies performed in human blood serum show the correct assembling of the probe butwith a reduction of limit of detection (up to∼1 nM). This liposome signal amplification strategy could be used not only for thedetection of DNA but also for other nucleic acids (mRNA; microRNA) that are difficult to be quantified by currently availableprotocols