Universal liposomes: preparation and usage for the detection of mRNA

Edwards, Katie A.; Curtis, Katherine L.; Sailor, Jessica L.; Baeumner, Antje J.
July 2008
Analytical & Bioanalytical Chemistry;Jul2008, Vol. 391 Issue 5, p1689
Academic Journal
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.


Related Articles

  • Amplification-Free Detection of Cryptosporidium parvum Nucleic Acids with the Use of DNA/RNA-Directed Gold Nanoparticle Assemblies. Weigum, S. E.; Castellanos-Gonzalez, A.; White Jr., A. C.; Richards-Kortum, R. // Journal of Parasitology;Oct2013, Vol. 99 Issue 5, p923 

    This study describes the development and evaluation of an amplification-free molecular assay for detection of Cryptosporidium parvum oocysts. The assay employed a pair of oligonucleotide-functionalized gold nanoparticle (AuNP) probes that were complementary to adjacent sequences on C. parvum 18s...

  • From Media to Molecules: New Approaches to the Detection of Micro-organisms in Water. Fricker, C.R. // Water, Air & Soil Pollution;Oct2000, Vol. 123 Issue 1-4, p35 

    Present methods for the detection of micro-organisms in the environment are slow, inefficient and often unreliable. Alternative approaches which are reliable and rapid, enabling results to be obtained within one working day, are required. The development of molecular techniques, in particular in...

  • An evaluation of primers amplifying DNA targets for the detection of Cryptosporidium spp. using C. parvum HNJ-1 Japanese isolate in water samples. Anna Leetz; Isaia Sotiriadou; Jerry Ongerth; Panagiotis Karanis // Parasitology Research;Sep2007, Vol. 101 Issue 4, p951 

    Abstract  The performance of polymerase chain reaction (PCR) procedures for the detection of Cryptosporidium parvum HNJ-1 strain (genotype II) oocysts purified from mice using published protocols was evaluated. Oocysts were concentrated from fecal samples of infected severe...

  • Simulating the XOR Gates Based on the Induced Hairpin Formation. Wenbin Liu; Xiangou Zhu; Xianghong Wang; Zhixiang Yin; Shudong Wang // Current Nanoscience;Feb2008, Vol. 4 Issue 1, p108 

    The hairpin structure of DNA molecules have been widely employed for a variety of biosensors and nanoscale molecular assembly applications. For example, the commonly known molecular beacons can report the presence of specific nucleic acids in homogeneous solutions with high accuracy. Recently,...

  • Biosensors for the detection of waterborne pathogens. Connelly, John; Baeumner, Antje // Analytical & Bioanalytical Chemistry;Feb2012, Vol. 402 Issue 1, p117 

    Waterborne bacterial, viral and parasitic pathogens are a global health concern and their rapid and specific detection in contaminated potable water is of utmost importance. Biosensors using a variety of biorecognition molecules and transduction methodologies have been reported, and have the...

  • Multi-analyte single-membrane biosensor for the serotype-specific detection of Dengue virus. Zaytseva, Natalya V.; Montagna, Richard A.; Eun Mi Lee; Baeumner, Antje J. // Analytical & Bioanalytical Chemistry;Sep2004, Vol. 380 Issue 1, p46 

    A multi-analyte biosensor based on nucleic acid hybridization and liposome signal amplification was developed for the rapid serotype-specific detection of Dengue virus. After RNA amplification, detection of Dengue virus specific serotypes can be accomplished using a single analysis within 25...

  • Immunofluorescence Assay and PCR Analysis of Cryptosporidium Oocysts and Species From Human Feacal Specimens. Vejdani, Mehdi; Mansour, Rezaei; Hamzavi, Yezdan; Vejdani, Sina; Nazeri, Naser; Michaeli, Ali // Jundishapur Journal of Microbiology;Jun2014, Vol. 7 Issue 6, p1 

    Background: Cryptosporidium spp. is a widespread protozoan parasite involving humans and animals. Objectives: This study was carried out to evaluate the immunofluorescence assay (IFA) and PCR results for more accurate diagnosis of faecal specimens. Patients and Methods: Forty six faecal human...

  • Complex approach to investigation of the mechanisms of action of antimicrobial and antiviral drugs. Bykov, V.; Dubinskaya, V.; Rebrov, L.; Mineeva, M.; Skuridin, S.; Evdokimov, Yu. // Pharmaceutical Chemistry Journal;Mar2008, Vol. 42 Issue 3, p105 

    Enzyme and biosensor test systems have been used in vitro to study the possible mechanisms of action of antimicrobial and antiviral drugs. It is established that compounds belonging to these groups of drugs may cause various modes of damage in the DNA secondary structure, possess complex-forming...

  • Biosensors for environmental pollutants and food contaminants. Baeumner, Antje J. // Analytical & Bioanalytical Chemistry;Oct2003, Vol. 377 Issue 3, p434 

    This review article provides an overview of the most recent literature on biosensors for environmental pollutants and food contaminants. Due to the large number of publications, only papers published between 2000 and January 2003 were considered. Also, while not all of the published literature...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics