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

Abstract

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.

The Application of Nanotechnology for Quantification of Circulating Tumour DNA in Liquid Biopsies: A Systematic Review

“…Multiple categories were assigned to each record, where applicable. A few technologies were not captured by these categories, such as liposomes [90]. The readout types used were: absorbance/colour, electrochemical, fluorescence, SPR/Raman and other.…”

Nucleic Acid‐Modified Liposome: Construction Methods and Biological Applications

“…[ 40 ] This method usually uses one DNA strand anchored on magnetic beads as a anchor chain, and the other DNA strand was attached on the liposome as a reporter strand ( Figure a). [ 41 ] When the target exists in the system, the sensor can capture the target chain to form a liposome‐nucleic acid chain‐magnetic bead composite structure, which is then concentrated by magnets for sensitive detection of targets. The detection limit of this system is relatively low.…”

“…Reproduced with permission. [ 41b ] Copyright 2005, American Chemical Society. b) A turn‐on biosensor is constructed by using SSB to combine an aptamer with one end modified nano‐gold and a liposome carrying quantum dots.…”

Liposome-Based Bioassays

“…The most used dyes encapsulated in liposomes are sulforhodamine and carboxyfluorescein [ 53 ]. One of the most recently proposed assays of this class used 18 bp long probes bound to streptavidin-coated magnetic beads through a biotin moiety [ 57 ]. Liposomes were previously conjugated with a DNA probe by means of a cholesterol moiety, able to intercalate in the phospholipid bilayer of the vesicle.…”

“…Upon washing out the unbound liposomes, the bound ones are quantitatively assessed upon the release of their fluorescent payload. Figure inspired by [ 57 ].…”