Optimization of DNA-tagged dye-encapsulating liposomes for lateral-flow assays based on sandwich hybridization

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.

Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey

“…A combination of antigen-antibody interaction and specific tagged doubled-stranded amplicon (ds-amplicon) detection after PCR is also possible, and is called "nucleic acid lateral flow immunoassay" (NALFIA) [39,40,42,69]. Specific nucleic acid hybridisation of amplicons with immobilised complementary probes is another option and is called "nucleic acid lateral flow assay" (NALF) [16,41]. A selection of published LFA applications with the method applied and the sensitivity is presented in Tables 1, 2, 3 and 4; these were mainly published in the last decade.…”

“…4b) [16]; (2) nanoparticle-labelled reporter probe and bovine serum albumin labelled capture probe, immobilised through passive adsorption, ss-amplicon hybridises with complementary reporter and capture probes (Fig. 4c) [70]; (3) nanoparticle-labelled reporter probe; capture probe is immobilised at the test line through passive adsorption, ss-amplicon hybridises with complementary reporter and capture probes (Fig.…”

“…nylon [73] (Millipore, Pall Biosciences), polyethersulfone [15,16] (Pall Biosciences or Nalgene-Nunc), polyethylene [51] (Porex) or fused silica (Fusion5, Whatman), but these are used less often. A slight tendency to use polyethersulfone material was observed for the NALF format [15,16]. Important are not only the capillary forces of the carrier material, but also the ease of binding and immobilising proteins necessary for sequential selection, reaction and detection.…”

“…Lateral flow (immuno)assay is a technology that is currently widely applied [5][6][7][8][9][10][11][12][13][14][15][16]; however, to our knowledge no in-depth survey of recent literature is available. Here we present a survey of development reports presented in the scientific literature, mainly during the last decade, where a selection of those applications is mentioned that are real one-step dry reagent assays with coloured or luminescent response.…”

The Application of Lateral Flow Immunoassay in Point of Care Testing: A Review

“…So the signal intensity could be amplified greatly. Some researchers utilized lipidosome as labels which offer higher sensitivity owning to its strong plasticity [50], ability to set multiple antigens and antibodies in the membrane and to contain plenty of indicator in water phase space inside the membrane. Gred, et al used europium microparticles as labels in immunochromatography system.…”