Universal liposomes: preparation and usage for the detection of mRNA.
Dye-encapsulating liposomes can serve as signaling reagents in biosensors and biochemical assays in place of enzymes or fluorophores. Detailed here is the use and preparation of streptavidin-coupled liposomes which offer a universal approach to biotinylated target detection. The universal approach provides two advantages, i.e. only one type of liposome is necessary despite varying target and probe sequences and the hybridization event can take place in the absence of potential steric hindrance occurring from liposomes directly conjugated to probes. One objective of this work was to optimize the one-step conjugation of SRB-encapsulating liposomes to streptavidin using EDC. Liposome, EDC, streptavidin concentrations, and reaction times were varied. The optimal coupling conditions were found to be an EDC:carboxylated lipid:streptavidin molar ratio of 600:120:1 and a reaction time of 15 min. The second goal was to utilize these liposomes in sandwich hybridization microtiter plate-based assays using biotinylated reported probes as biorecognition elements. The assay was optimized in terms of probe spacer length, probe concentration, liposome concentration, and streptavidin coverage. Subsequently, the optimized protocol was applied to the detection of DNA and RNA sequences. A detection limit of 1.7 pmol L(-1) and an assay range spanning four orders of magnitude (5 pmol L(-1)-50 nmol L(-1)) with a coefficient of variation </=5.8% was found for synthetic DNA. For synthetic RNA the LOQ was half that of synthetic DNA. A comparison was made to alkaline phosphatase-conjugated streptavidin for detection which yielded a limit of quantitation approximately 80 times higher than that for liposomes in the same system. Thus, liposomes and the optimized sandwich hybridization method are well suited for detecting single-stranded nucleic acid sequences and compares favorably to other sandwich hybridization schemes recently described in the literature. The assay was then used successfully for the clear detection of mRNA amplified by nucleic acid sequence-based amplification (NASBA) isolated from as little as one Cryptosporidium parvum oocyst. The detection of mRNA from oocysts isolated from various water sample types using immunomagnetic separation was also assessed. Finally, to prove the wider applicability and sensitivity of this universal method, RNA amplified from the atxA gene of Bacillus anthracis was detected when the input to the preceding NASBA reaction was as low as 1.2 pg. This highly sensitive liposome-based microtiter plate assay is therefore a platform technology allowing for high throughput and wide availability for routine clinical and environmental laboratory applications.
Edwards
Katie A
KA
Biological and Environmental Engineering, Cornell University, 145 Riley-Robb Hall, Ithaca, New York, 14853, USA.
Curtis
Katherine L
KL
Sailor
Jessica L
JL
Baeumner
Antje J
AJ
D000818:Animals; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009696:Nucleic Acids / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D008081:Liposomes; D009696:Nucleic Acids; D012333:RNA, Messenger; D012321:DNA-Directed RNA Polymerases; D019793:Fluorescein