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Nov 4, 2011

Nanolipid Vesicle Platform Developed that Uses Subattoliter Volumes for Screening Reactions

Nanolipid Vesicle Platform Developed that Uses Subattoliter Volumes for Screening Reactions

Approach devised by Danish team involves fusion of nanovesicles carrying target and cargo reactants.[AVAVA - Fotolia.com]

  • Scientists report on a nanoscale lipid vesicle-based platform that allows millions of biochemical reactions to be tested simultaneously on a single chip, using subattoliter volumes of reagents. The technique, developed by a team at the University of Copenhagen’s Bionanotechnology and Nanomedicine Laboratory in the Department of Neuroscience and Pharmacology, is based on the fusion of small unilaminar vesicles (SUVs) encasing volumes of reactants as small as 10-19 liters.

    Reporting in Nature Nanotechnology, Dimitrios Stamou, Ph.D., and colleagues say the platform is highly reproducible, and enabled them to carry out and detect a million individual chemical reactions per cm2 in just a few minutes. They describe the system in a paper titled “Mixing subattolitre volumes in a quantitative and highly parallel manner with soft matter nanofluidics.”

    Working with ultrasmall volumes of reactants can increase the throughput and complexity of screening assays and significantly reduce reagent use, but while current platforms based on microfabricated silicon and plastic can provide reliable fluidic devices, they can’t generally handle total volumes smaller than about 1 x 10-12 liters, the authors explain. In contrast, self-assembled "soft matter" nanocontainers could improve miniaturization and biocompatibility, but working with such small dimensions presents a real challenge.

    While the use of high-density surface-based arrays of autonomous SUVs for characterizing single vesicles has been demonstrated, to date no method has been developed to trigger reactions within the self-enclosed volume of individually immobilized SUVs, they report.

    The Copenhagen team’s approach is based on fusing two sets of SUVs containing reactant molecules. Random arrays of subattoliter-sized nanocontainers are first generated by immobilizing one set of charged SUVs (the target reactors) on functionalized glass surfaces. A second set of oppositely charged SUVs encasing the complementary reactants (the cargo reactors) is then generated and diffused over the immobilized target reactors. The oppositely charged SUVs fuse, their contents mix and react, and the products can be detected by standard techniques such as fluorescence.

    The team initially confirmed the feasibility of the technique by fluorescently tagging the SUV lipids and their reacting contents. They found that full lipid mixing occurred in less than 65 ms, and based on previous kinetic data, content mixing was expected to take place within a few tens of microseconds.

    In a proof of concept experiment designed to test the platform for screening biochemical reactions, the researchers loaded target and cargo reactors with alkaline phosphatase and a profluorescent substrate (fluorescein diphosphate, FDP), respectively. The reaction between these two molecules leads to FDP hydrolysis and formation of the fluorescent product (fluorescein). Tracking over 1,900 target reactors throughout the process of incubation, the team confirmed that product was generated in some 88% of the reactors. Repeating the experiment also demonstrated the reproducibility of the technique: each time the percentage of active reactors was about 80–90%.

    The system was then applied to test a range of reaction types, including the peroxidise-catalyzed formation of resorufin by reaction with Amplex Red, and the activity of Thermomyces lanuginosus lipase (TLL), a membrane-dependent lipase that is activated on binding to the inner lipid walls of the reactor. This combined data demonstrated the potential of the platform for operating diverse biochemical reactions, the authors state.

    They next tested whether there was any leakage associated with SUV fusion, by loading the immobilized target reactors with a water-soluble fluoscent molecule (Alexa 488 hydrazide), and monitoring whether there was any leakage during cargo-target reactor fusion. Encouragingly, the results indicated that no leakage occurred during content mixing and reaction.

    Finally, the team optimized construction of the lipid vesicles so that after fusion of one cargo SUV to its target SUV, the target would undergo minimal neutralization of charge, and thus allow subsequent fusions to occur. To achieve this they decreased the total lipid mass ratio of the cargo to target reactor, by extruding them at 50 nm and 400 nm, respectively. This modification allowed at least a third of the target reactors to undergo two rounds of fusion, and some underwent three or even four.

    The authors claim the platform represents one of the most advanced examples of functional nanoscale self-assembly with three-dimensional components, and will provide a foundation for further modifications to increase its utility even further. Such modifications could include developing high-density microarrays of single nanoreactors, using complementary DNA oligos to direct the fusion of specific cargo/target reaction pairs, or applying in situ sequencing technologies to barcode the products of random reactions.  “The development of such ultrasmall-volume fluidic platforms will lead to novel ways to implement the simultaneous screening of biochemical properties, molecular function, or confined chemical reactions over millions of samples while consuming total reagent volumes of a few picolitres,” they conclude.


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