0508964 - ÚPT 2020 RIV US eng J - Journal Article
Buryška, T. - Vašina, M. - Gielen, F. - Vaňáček, P. - van Vliet, L. - Ježek, Jan - Pilát, Zdeněk - Zemánek, Pavel - Damborský, J. - Hollfelder, F. - Prokop, Z.
Controlled Oil/Water Partitioning of Hydrophobic Substrates Extending the Bioanalytical Applications of Droplet-Based Microfluidics.
Analytical Chemistry. Roč. 91, č. 15 (2019), s. 10008-10015. ISSN 0003-2700. E-ISSN 1520-6882
R&D Projects: GA ČR(CZ) GA16-07965S; GA MŠMT ED0017/01/01
Institutional support: RVO:68081731
Keywords : enzyme-kinetics * platform * chip
OECD category: Optics (including laser optics and quantum optics)
Impact factor: 6.785, year: 2019 ; AIS: 1.411, rok: 2019
Method of publishing: Limited access
Result website:
https://pubs.acs.org/doi/10.1021/acs.analchem.9b01839DOI: https://doi.org/10.1021/acs.analchem.9b01839

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.
Permanent Link: http://hdl.handle.net/11104/0299794