Browsing by Subject "Single enzyme molecule activity"

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    Development of a microfluidic, segmented-flow, single molecule, enzyme activity assay and improvement of separation efficiency of basic proteins by application of a water- proofing agent as a coating in capillary electrophoresis
    (2012-08) Castro Barahona, Eric Rigoberto
    A novel, microfluidic platform for segmented flow assays has been developed using commercially available Teflon tubing and PEEK connectors. Such a system can be used to generate arrays of nano to pico liter sized droplets separated from each other by plugs of a fluourous solvent. Each droplet becomes an individual reaction vessel suitable for high-throughput applications. We have applied this method to the development of a single enzyme molecule fluorescence assay. Characterization of the droplet generation platform was done with the use of a 100 μm ID PEEK T-junction connector. When two immiscible streams, such as water and a fluorous solvent, meet at the T-junction an array of aqueous droplets separated by plugs of the solvent is generated inside the Teflon tubing. Experiments have shown that, like previous microfabricated segmented flow devices, our system can control the size of the droplets generated solely by changing the ratio of the flow rates of the two phases. Using this approach droplets can be produced with good reproducibility (better than 6% in all cases and better than 3% in most) over a wide range of flow rates. Rates of droplet generation of 10.37 ± 0.17 drops/s are easily achieved for good high-throughput potential. Fast on-line mixing of reagents and long term droplet stability of up to 7 days has also been demonstrated. The discovery of the heterogeneity of enzyme molecules with respect to activity has resulted in the development of a variety of single enzyme molecule assays, with the aim of investigating the prevalence and origin of this phenomenon. The segmented flow platform we have developed is well suited to the application of single enzyme assays. It has the advantage of high-throughput, as well as ease of fabrication compared to PDMS or silica based devices and elimination of exposure of the enzyme analyte to the walls of the channel or well. A segmented flow, single molecule assay has been developed for the enzyme alkaline phosphatase (AP). Single AP molecules were sequestered inside 100 pL droplets generated in a PEEK tee and stored in a length of 50 μm ID Teflon tubing. The droplet array was allowed to incubate for a suitable time period, during which the AP molecules converted the weakly fluorescent substrate AttoPhos® into a strongly fluorescent product. AP molecules were found, as in previous studies, to display heterogenous activity with up to a 9-fold difference between individual enzymes In the last section of this work we have used the commercial glass treatment Aquapel as a capillary wall coating agent to reduce protein absorption in capillary electrophoresis (CE). Due to their large number of potential sites for interactions with the fused-silica wall, protein separations with CE can often be difficult. For this reason, much effort is expended on the development on wall coating agents for the prevention of such interactions. Aquapel is a fluorous polymer used commercially to render glass surfaces hydrophobic. The efficacy of the coating was investigated using a suite of three basic proteins: lysozyme, cytochrome c and α-chymotrypsinogen. Separation efficiencies of up to 130,000 theoretical plates were achieved over a pH range of 4.0 to 7.0, a significant improvement over bare fused silica capillary. Electroosmotic flow (EOF) was reduced by the Aquapel coating but not entirely suppressed. The stability of the coating was also examined. 62 protein injections were performed over a two day period during which analyte migration times varied by less than 3.5%. Due to the ease of application and low cost, coating with Aquapel is an attractive alternative to available capillary coatings.