Femtomole-Scale High-Throughput Screening of Protein Ligands with Droplet-Based Thermal Shift Assay

Analytical Chemistry
Wen-Wen LiuQun Fang

Abstract

There is a great demand to measure protein-ligand interactions in rapid and low cost way. Here, we developed a microfluidic droplet-based thermal shift assay (dTSA) system for high-throughput screening of small-molecule protein ligands. The system is composed of a nanoliter droplet array chip, a microfluidic droplet robot, and a real-time fluorescence detection system. Total 324 assays could be performed in parallel in a single chip with an 18 × 18 droplet array. The consumption of dTSA for each protein or ligand sample was only 5 nL (femtomole scale), which is significantly reduced by over 3 orders of magnitude compared with those in 96- or 384-well plate-based systems. We also observed the implementation of TSA in nanoliter droplet format could substantially improve assay precision with relative standard deviation (RSD) of 0.2% (n = 50), which can be ascribed to the enhanced thermal conduction in small volume reactors. The dTSA system was optimized by studying the effect of droplet volumes, as well as protein and fluorescent dye (SYPRO Orange) concentrations. To demonstrate its potential in drug discovery, we applied the dTSA system in screening inhibitors of human thrombin with a commercial library containing 100 different s...Continue Reading

References

Feb 19, 2000·Current Opinion in Biotechnology·R L Rich, D G Myszka
Jan 12, 2002·Current Opinion in Structural Biology·S Leavitt, E Freire
Jan 15, 2002·Journal of Biomolecular Screening·M W PantolianoF R Salemme
Apr 10, 2002·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·David S Hage
Jun 19, 2002·Blood·Kathleen E BrummelKenneth G Mann
Apr 25, 2003·Journal of Pharmaceutical Sciences·Michael J BankerJohn A Williams
May 19, 2004·Nucleic Acids Research·Dagmar KlostermeierJames R Williamson
Aug 11, 2004·Analytical Biochemistry·Mei-Chu LoGeorge Ellestad
Mar 7, 2006·Nature Reviews. Drug Discovery·Petra S Dittrich, Andreas Manz
Feb 1, 1958·Proceedings of the National Academy of Sciences of the United States of America·D E Koshland
Aug 2, 2006·Molecular BioSystems·Mahesh UttamchandaniShao Q Yao
Sep 12, 2006·Analytical Biochemistry·Ulrika B EricssonPär Nordlund
Oct 13, 2006·Proceedings of the National Academy of Sciences of the United States of America·Masoud VedadiAled M Edwards
Sep 6, 2008·Nature Protocols·Hongyan SunShao Q Yao
Feb 13, 2009·Journal of Biomolecular Screening·Fang YiLynne Regan
Mar 19, 2009·Journal of the American Chemical Society·Jason J LavinderThomas J Magliery
May 14, 2009·Accounts of Chemical Research·Mahesh UttamchandaniShao Q Yao
Jun 6, 2009·Chembiochem : a European Journal of Chemical Biology·Monpichar Srisa-ArtAndrew J deMello
Mar 5, 2011·Methods in Enzymology·James K Kranz, Celine Schalk-Hihi
Dec 14, 2011·Journal of Medicinal Chemistry·Hannah J MapleJohn Crosby
Nov 14, 2012·Journal of Pharmaceutical and Biomedical Analysis·Jakob WallnerKarola Vorauer-Uhl
Sep 11, 2014·Analytical Chemistry·Lih Feng CheowWilliam F Burkholder

❮ Previous
Next ❯

Related Concepts

Related Feeds

Cardiac Conduction System

The cardiac conduction system is a specialized tract of myocardial cells responsible for maintaining normal cardiac rhythm. Discover the latest research on the cardiac conduction system here.