A novel luminescent biosensor for rapid monitoring of IP3 by split-luciferase complementary assay

Inositol 1,4,5-trisphosphate (IP3) is a crucial second messenger that regulates complicated signaling processes in various physiological events. Alteration in its content has been observed in many diseases. Hence, development of a high-throughput screening system to monitor temporal changes of IP3 is essential for screening of new potential therapeutic compounds. Toward a simple, sensitive and rapid method for measuring IP3, we describe the development and application of a novel biosensor based on luciferase fragment assisted complementation strategy, which converts the ligand-induced conformational changes to light. Designed sensor comprising the IP3-binding core domain of IP3-receptor fused between complementary non-functional fragments of firefly luciferase allows direct detection of IP3 in presence of luciferin substrate both in cell lysate and in living cells. According to the result presented in this manuscript, the screening time was very fast and maximum response was obtained up to 11-fold higher than untreated cells. Moreover, the designed biosensor was able to monitor release of IP3 upon induction by different inducers like Bradykinin and ATP. The current biosensor not only provides a specific IP3 detector in vitro but also facilitates monitoring of the response of IP3 in living organisms. (C) 2012 Elsevier B.V. All rights reserved.