dc.contributor.author | Buryska, T | |
dc.contributor.author | Vasina, M | |
dc.contributor.author | Gielen, F | |
dc.contributor.author | Vanacek, P | |
dc.contributor.author | van Vliet, L | |
dc.contributor.author | Jezek, J | |
dc.contributor.author | Pilat, Z | |
dc.contributor.author | Zemanek, P | |
dc.contributor.author | Damborsky, J | |
dc.contributor.author | Hollfelder, F | |
dc.contributor.author | Prokop, Z | |
dc.date.accessioned | 2019-07-16T13:38:56Z | |
dc.date.issued | 2019-07-10 | |
dc.description.abstract | 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 °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. | en_GB |
dc.description.sponsorship | Czech Science Foundation | en_GB |
dc.description.sponsorship | Ministry of Education Youths and Sports of the Czech Republic | en_GB |
dc.description.sponsorship | European Commission | en_GB |
dc.description.sponsorship | ERC | en_GB |
dc.identifier.citation | Published online 26 June 2019 | en_GB |
dc.identifier.doi | 10.1021/acs.analchem.9b01839 | |
dc.identifier.grantnumber | GA16-07965S | en_GB |
dc.identifier.grantnumber | LQ1605 LO1214 | en_GB |
dc.identifier.grantnumber | CZ.02.1.01/0.0/0.0/16_026/0008451 | en_GB |
dc.identifier.grantnumber | CZ.02.1.01/0.0/0.0/16_019/0000868 | en_GB |
dc.identifier.grantnumber | LM2015051 | en_GB |
dc.identifier.grantnumber | CZ.02.1.01/0.0/0.0/16_013/0001761 | en_GB |
dc.identifier.grantnumber | 720776 | en_GB |
dc.identifier.grantnumber | CZ.1.05/2.1.00/01.0017 | en_GB |
dc.identifier.grantnumber | 695669 | en_GB |
dc.identifier.uri | http://hdl.handle.net/10871/37999 | |
dc.language.iso | en | en_GB |
dc.publisher | American Chemical Society | en_GB |
dc.relation.url | https://www.ncbi.nlm.nih.gov/pubmed/31240908 | en_GB |
dc.rights.embargoreason | Under embargo until 10 July 2020 in compliance with publisher policy. | en_GB |
dc.rights | © 2019 American Chemical Society. | en_GB |
dc.title | Controlled Oil/Water Partitioning of Hydrophobic Substrates Extending the Bioanalytical Applications of Droplet-Based Microfluidics. | en_GB |
dc.type | Article | en_GB |
dc.date.available | 2019-07-16T13:38:56Z | |
dc.identifier.issn | 0003-2700 | |
exeter.place-of-publication | United States | en_GB |
dc.description | This is the author accepted manuscript. The final version is available from American Chemical Society via the DOI in this record. | en_GB |
dc.identifier.journal | Analytical Chemistry | en_GB |
dc.rights.uri | http://www.rioxx.net/licenses/all-rights-reserved | en_GB |
dcterms.dateAccepted | 2019-06-26 | |
rioxxterms.version | AM | en_GB |
rioxxterms.licenseref.startdate | 2019-07-10 | |
rioxxterms.type | Journal Article/Review | en_GB |
refterms.dateFCD | 2019-07-16T13:32:18Z | |
refterms.versionFCD | AM | |
refterms.panel | B | en_GB |