Integrated nanoreactor system: triggering (bio-)chemical reactions in single vesicles

This thesis describes a method that allows the production in parallel of nano-reactor systems whose function is controlled by a remote stimulus. The reactors comprise a nested system of lipid vesicles part of which release their content during a thermotropic phase transition. The integration of all components (substrate and enzyme for instance) in a single element eliminates the need for external manipulation/intervention and therefore renders this nanoscopic system entirely autonomous. The smallest attainable size for vesicles (diameter 20 nm, volume 10-21 L) puts a lower limit to the total volume of the device of about 10-18 L making it ideally suited for manipulating interacting partners at the single molecule level. The proof of principle of individual reactors immobilized on glass is first characterized using confocal microscopy and a fluorescent dye that reports dilution during the release. In a further step, enzymatic reactions were performed and recorded by fluorescence microscopy down to single vesicle level. Initial reaction rates were evaluated and compared between several containers showing a dependency with encapsulated substrate concentration. Using vesicles of several different lipid phase transition temperatures, the process was extended to perform 2 consecutive enzymatic reactions. In addition to confined enzymatic reactions, the system was characterized by fluorescence correlation spectroscopy providing the determination of the concentration of fluorophores and small vesicles incorporated inside individual larger containers. Combination of this method with vesicle microarray technology will permit the simultaneous observation and quantitative analysis of confined (bio)chemical reactions in millions of separated reactors and may find applications as high-throughput screening of single enzyme reaction system. The possibility to perform multiple consecutive biochemical reactions may permit to use this system as artificial cells.

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