Optimization of DNA-tagged dye-encapsulating liposomes for lateral-flow assays based on sandwich hybridization |
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Authors: | Katie A Edwards Antje J Baeumner |
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Institution: | (1) Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA |
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Abstract: | A novel protocol for the synthesis of dye-encapsulating liposomes tagged with DNA oligonucleotides at their outer surface
was developed. These liposomes were optimized for use as signal enhancement agents in lateral-flow sandwich-hybridization
assays for the detection of single-stranded RNA and DNA sequences. Liposomes were synthesized using the reverse-phase evaporation
method and tagged with oligonucleotides by adding cholesteryl-modified DNA probes to the initial lipid mixture. This resulted
in a greatly simplified protocol that provided excellent control of the probe coverage on the liposomes and cut the preparation
time from 16 hours to just 6 hours. Liposomes were prepared using probe concentrations ranging from 0.00077 to 0.152 mol%
of the total lipid, several hydrophobic and polyethylene glycol-based spacers between the cholesteryl anchor and the probe,
and liposome diameters ranging from 208 nm to 365 nm. The liposomes were characterized by dynamic light scattering, visible
spectroscopy, and fluorescence spectroscopy. Their signal enhancement functionality was compared by using them in lateral-flow
optical biosensors for the detection of single-stranded DNA sequences. In these assays, an optimal reporter probe concentration
of 0.013 mol%, liposome diameter of 315 nm, and liposome optical density of 0.4–0.6 at 532 nm were found. The spacer length
between the cholesteryl anchor and the probe showed no significant effect on the signals in the lateral-flow assays. The results
presented here provide important data for the general use of liposomes as labels in analytical assays, with specific emphasis
on nucleic acid detection via lateral flow assays. |
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Keywords: | Liposomes DNA Sandwich hybridization Conjugation Detection Biosensor |
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