Enzyme Kinetic Studies in Droplet Microfluidic Device with Fluorescence Detection
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Rok publikování | 2024 |
Druh | Konferenční abstrakty |
Citace | |
Popis |
Enzyme kinetics is important for a wide range of disciplines. As many enzymes play their role in human diseases, they are potential therapeutical drug targets. On the other hand, biotechnology field is other emerging area utilizing kinetic data in process optimization. However today multiple well plate format offers high degree of automation it is still characterized by significant consumption of all reagents. Droplet microfluidics (DM) enables the translation of chemical and biological assays to scales and rates unachievable in conventional laboratory workflows. Since the droplets are isolated and protected by the oil (water-in-oil concept), they can be individually manipulated and virtually regarded as separate microreactors. Compared to the conventional technologies, microfluidics offers several advantages such as reduced reagent consumption, shorter analyses times, and rapid production of data in given time. Working in minuscule volumes presents new possibilities in sample manipulation as well as challenges. The "hearth" of the DM components is typically polydimethylsiloxane chip with integrated droplet generator, incubation channel and other functional elements, connected to precise syringe pumps. Many different detection techniques were reported in combination with DM [1]. Laser induced fluorescence is one of them being often employed. In this study we monitored enzyme reaction of ß-galactosidase using synthetic fluorogenic substrate. First, chip fabrication process, inner surface modification as well as water-oil phase ratio and flow rates were optimized as all these parameters have significant impact on uniform-sized droplets generation and repeatability. The optimized setup allowed on-line substrate concentration variation by setting corresponding flow rates of individual reaction components. Finally, enzyme activity of ß-galactosidase, which is an important enzyme used in biotechnology and bioanalysis was measured with its fluorogenic substrate in setup which allowed to measure the reaction rate simply by focusing on different distances in the incubation channel, corresponding to different reaction times. Acknowledgement This study was supported by grant CZ-02.2.69/0.0/0.0/16_018/0002605. Special thanks belong to Dr. David Kovář and prof. Zbyněk Prokop for the help with chip fabrication. References [1] Y. Zhu, Q. Fang, Analytical detection techniques for droplet microfluidics—A review, Anal. Chim. Acta 787 (2013) 24–35. |
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