Enzyme kinetics and inhibition studies on microfluidic chip with on-line variation of analyte concentration
| Název česky | Studium enzymové kinetiky a inhibice na mikrofluidním čipu s on-line změnou aplikované koncentrace analytu |
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| Rok publikování | 2025 |
| Druh | Konferenční abstrakty |
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| Popis | Microfluidic devices have emerged as an alternative to conventional, larger-scale analytical methods to minimize the consumption of samples and consumables while obtaining relevant data. Microfluidic platforms also offer greater variability and control over the reaction process by incorporating additional functional components into the chip. In this study, we used a polydimethylsiloxane chip bonded to a glass slide made in a laboratory. The chip structure incorporated the input of reaction components, a merging region, a droplet generator, a mixing undulating curve and a reaction-detection channel. The liquids were delivered by precise pumps, which enabled control over the final concentration of reagents in the reaction mixture. Droplets were passively generated at the perpendicular junction where the oil phase separated the reaction mixture into droplets due to shear and viscosity effects. The enzymatic reaction of ß-galactosidase, an important enzyme used in biotechnology and bioanalysis, was monitored using a fluorogenic lactose analogue as the substrate. The reaction product, fluorescein, was detected using fluorescence. This setup enabled online adjustment of the concentrations of the substrate and inhibitor by setting the corresponding flow rates of the reaction mixture components. Generating 400–500 droplets per minute, each with a volume of approximately 10 nL, provides a high level of information with minimal sample consumption. Furthermore, sensitive fluorescence detection and a high droplet generation rate enabled the rapid assessment of enzyme kinetics at initial reaction rates. Studying enzyme inhibition by various substances is important for understanding reaction mechanisms, as well as for biomedical and biotechnological applications. This approach can be used to screen candidate drugs, for example. |
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