DNA-oligonucleotide encapsulating liposomes as a secondary signal amplification means

A novel liposome-based signal amplification system was developed by encapsulating DNA oligonucleotides within antibody-tagged liposomes and subsequently detecting the oligonucleotide with dye-encapsulating liposomes for double signal enhancement. In this sandwich immunoassay, the model analyte, protective antigen protein from B. anthracis, was captured by one set of antibodies immobilized in microtiter plate wells and detected using a second antibody conjugated to oligonucleotide-encapsulating liposomes. Bound liposomes were lysed releasing the encapsulated fluorescein-tagged DNA 25-mer probe, which was then permitted to hybridize with its complementary sequence immobilized in a second plate. Finally, the amount of oligonucleotide was detected through the addition of anti-fluorescein antibody tagged dye-encapsulating liposomes. These secondary liposomes allowed for a approximately 400x lower LOD than detection of the fluorescein-labeled probe alone. Several aspects were investigated, including the encapsulation of various oligonucleotide concentrations within liposomes; oligonucleotide hybridization times and buffers; degree of anti-fluorescein antibody coverage on the liposomes; and immobilized anti-protective antigen antibody concentration. We found that the encapsulation efficiency increased with the starting oligonucleotide concentration. As many as 4000 DNA 25-mers were successfully entrapped in the liposome, and minimal leakage was observed over the course of 8 months. When used in the sandwich immunoassay, a limit of detection of 4.1 ng/mL protective antigen was observed with an upper limit of 5000 ng/mL. Due to the endless combination of DNA oligonucleotide sequences, this assay lends itself perfectly for multiplexing on the order of tens to hundreds of analytes.