2017 Volume 30 Issue 6 Pages 657-660
The spatiotemporal control of enzyme reactions based on the combined use of a caged substrate and local photoirradiation was proposed for use in the high-throughput screening of enzymes on a microwell array chip. An enzyme reaction is initiated immediately after the mixing of an enzyme and its substrate. Since, rapid injection or addition of a reagent to initiate the reaction with appropriate timing is required for the fine measurement of the reaction. However, it is usually very difficult to inject an additional reagent in micrometer-sized wells. The coupled reactions of pyruvate kinase and luciferase with caged adenosine diphosphate (caged ADP) were used as a model of enzyme reactions to be controlled by phototriggering. The coupled reactions were suppressed until photoirradiation and were initiated by the photoirradiation at 340-380 nm. The extent of the reactions was proved to be controlled by adjusting the photoirradiation dose; 500 mJ/cm2 was sufficient to saturate the extent of the triggered reactions with 0.1 mM caged ADP in 50 μL. Multistep measurements of enzyme reactions by reusing the same reaction solution were carried out by multistep phototriggering at fixed or various doses. The phototriggering technique enables the measurement of enzyme reactions in micrometer-sized microwells.