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Controlled Oil/Water Partitioning of Hydrophobic Substrates Extending the Bioanalytical Applications of Droplet-Based Microfluidics
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SYSNO ASEP 0508964 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Controlled Oil/Water Partitioning of Hydrophobic Substrates Extending the Bioanalytical Applications of Droplet-Based Microfluidics Author(s) Buryška, T. (CZ)
Vašina, M. (CZ)
Gielen, F. (GB)
Vaňáček, P. (CZ)
van Vliet, L. (GB)
Ježek, Jan (UPT-D) RID, ORCID, SAI
Pilát, Zdeněk (UPT-D) RID, SAI, ORCID
Zemánek, Pavel (UPT-D) RID, SAI, ORCID
Damborský, J. (CZ)
Hollfelder, F. (GB)
Prokop, Z. (CZ)Number of authors 11 Source Title Analytical Chemistry. - : American Chemical Society - ISSN 0003-2700
Roč. 91, č. 15 (2019), s. 10008-10015Number of pages 8 s. Publication form Print - P Language eng - English Country US - United States Keywords enzyme-kinetics ; platform ; chip Subject RIV BH - Optics, Masers, Lasers OECD category Optics (including laser optics and quantum optics) R&D Projects GA16-07965S GA ČR - Czech Science Foundation (CSF) ED0017/01/01 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) Method of publishing Limited access Institutional support UPT-D - RVO:68081731 UT WOS 000480499200092 EID SCOPUS 85071179907 DOI 10.1021/acs.analchem.9b01839 Annotation 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. Workplace Institute of Scientific Instruments Contact Martina Šillerová, sillerova@ISIBrno.Cz, Tel.: 541 514 178 Year of Publishing 2020 Electronic address https://pubs.acs.org/doi/10.1021/acs.analchem.9b01839
Number of the records: 1