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Controlled Oil/Water Partitioning of Hydrophobic Substrates Extending the Bioanalytical Applications of Droplet-Based Microfluidics

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BURYŠKA Tomáš VAŠINA Michal GIELEN Fabrice VAŇÁČEK Pavel VAN VLIET Liisa JEZEK Jan PILAT Zdenek ZEMÁNEK Pavel DAMBORSKÝ Jiří HOLLFELDER Florian PROKOP Zbyněk

Rok publikování 2019
Druh Článek v odborném periodiku
Časopis / Zdroj Analytical Chemistry
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Citace
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Doi http://dx.doi.org/10.1021/acs.analchem.9b01839
Klíčová slova ENZYME-KINETICS; PLATFORM; CHIP; COMPARTMENTS; SENSITIVITY; ABSORBENCY; EVOLUTION; TRANSPORT; ASSAYS; SPACE
Popis Functional annotation of novel proteins lags behind the number of sequences discovered by the next-generation sequencing. The throughput of conventional testing methods is far too low compared to sequencing; thus, experimental alternatives are needed. Microfluidics offer high throughput and reduced sample consumption as a tool to keep up with a sequence-based exploration of protein diversity. The most promising droplet-based systems have a significant limitation: leakage of hydrophobic compounds from water compartments to the carrier prevents their use with hydrophilic reagents. Here, we present a novel approach of substrate delivery into microfluidic droplets and apply it to high-throughput functional characterization of enzymes that convert hydrophobic substrates. Substrate delivery is based on the partitioning of hydrophobic chemicals between the oil and water phases. We applied a controlled distribution of 27 hydrophobic haloalkanes from oil to reaction water droplets to perform substrate specificity screening of eight model enzymes from the haloalkane dehalogenase family. This droplet-on-demand microfluidic system reduces the reaction volume 65 000-times and increases the analysis speed almost 100-fold compared to the classical test tube assay. Additionally, the microfluidic setup enables a convenient analysis of dependences of activity on the temperature in a range of 5 to 90 degrees C for a set of mesophilic and hyperstable enzyme variants. A high correlation between the microfluidic and test tube data supports the approach robustness. The precision is coupled to a considerable throughput of >20 000 reactions per day and will be especially useful for extending the scope of microfluidic applications for high-throughput analysis of reactions including compounds with limited water solubility.
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