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

The development of an innovative and easy way to run assays for the quantitative detection of DNA present in biological fluids (i.e., blood, urine, and saliva) is of great interest for early diagnosis (e.g., tumors) and personalized medicine. Herein, a new quantitative assay based on the use of highly sensitive carboxyfluorescein-loaded liposomes as signal amplification systems is reported. The method has been tested for the detection of low amounts of DNA sequences. The reported proof of concept exploits a target DNA molecule as a linker between two complementary oligonucleotides. One oligonucleotide is biotinylated at its 3' end and binds to streptavidin-coupled magnetic beads, whereas the other one is conjugated to a cholesterol molecule incorporated in the phospholipidic bilayer of the fluorescent liposomes. In the presence of the target fragment, the correct formation of a construct takes place as witnessed by a strong fluorescence signal, amplified by dissolving lipidic nanoparticles with Triton X-100. The system is able to detect specific nucleotide sequences with a very low detection threshold of target DNA (tens of picomolar). The assay allows the detection of both single- and double-stranded DNA. Studies performed in human blood serum show the correct assembling of the probe but with a reduction of limit of detection (up to ∼1 nM). This liposome signal amplification strategy could be used not only for the detection of DNA but also for other nucleic acids (mRNA; microRNA) that are difficult to be quantified by currently available protocols.