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Adaptation of a Microfluidic qPCR System for Enzyme Kinetic Studies.

ABSTRACT: Microfluidic platforms offer a drastic increase in throughput while minimizing sample usage and hands-on time, which make them important tools for large-scale biological studies. A range of such systems have been developed for enzyme activity studies, although their complexity largely hinders their application to the wider scientific community. Here, we present adaptation of an easy-to-use commercial microfluidic qPCR system for performing enzyme kinetic studies. We demonstrate the functionality of the Fluidigm Biomark HD system (the Fluidigm system) by determining the kinetic properties of three oxidases in a resorufin-based fluorescence assay. The results obtained in the microfluidic system proved reproducible and comparable to the ones obtained in a standard microplate-based assay. With a wide range of easy-to-use, off-the-shelf components, the microfluidic system presents itself as a simple and customizable platform for high-throughput enzyme activity studies.

SUBMITTER: Rembeza E

PROVIDER: S-EPMC7841792 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Adaptation of a Microfluidic qPCR System for Enzyme Kinetic Studies.

Rembeza Elzbieta E Engqvist Martin K M MKM

ACS omega 20210113 3

Microfluidic platforms offer a drastic increase in throughput while minimizing sample usage and hands-on time, which make them important tools for large-scale biological studies. A range of such systems have been developed for enzyme activity studies, although their complexity largely hinders their application to the wider scientific community. Here, we present adaptation of an easy-to-use commercial microfluidic qPCR system for performing enzyme kinetic studies. We demonstrate the functionality ...[more]

PMID: 33521438

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Project description:Stability and biological activity of proteins is highly dependent on their physicochemical environment. The development of realistic models of biological systems necessitates quantitative information on the response to changes of external conditions like pH, salinity and concentrations of substrates and allosteric modulators. Changes in just a few variable parameters rapidly lead to large numbers of experimental conditions, which go beyond the experimental capacity of most research groups. We implemented a computer-aided experimenting framework ("robot lab assistant") that allows us to parameterize abstract, human-readable descriptions of micro-plate based experiments with variable parameters and execute them on a conventional 8 channel liquid handling robot fitted with a sensitive plate reader. A set of newly developed R-packages translates the instructions into machine commands, executes them, collects the data and processes it without user-interaction. By combining script-driven experimental planning, execution and data-analysis, our system can react to experimental outcomes autonomously, allowing outcome-based iterative experimental strategies. The framework was applied in a response-surface model based iterative optimization of buffer conditions and investigation of substrate, allosteric effector, pH and salt dependent activity profiles of pyruvate kinase (PYK). A diprotic model of enzyme kinetics was used to model the combined effects of changing pH and substrate concentrations. The 8 parameters of the model could be estimated from a single two-hour experiment using nonlinear least-squares regression. The model with the estimated parameters successfully predicted pH and PEP dependence of initial reaction rates, while the PEP concentration dependent shift of optimal pH could only be reproduced with a set of manually tweaked parameters. Differences between model-predictions and experimental observations at low pH suggest additional protonation-sites at the enzyme or substrates critical for enzymatic activity. The developed framework is a powerful tool to investigate enzyme reaction specifics and explore biological system behaviour in a wide range of experimental conditions.

Dataset Information

Adaptation of a Microfluidic qPCR System for Enzyme Kinetic Studies.

Publications

Adaptation of a Microfluidic qPCR System for Enzyme Kinetic Studies.

Similar Datasets

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