Developing of bioluminescent ratiometric biosensors for pH and divalent metals based on the engineering of the pH-sensing moiety of firefly luciferases

• Grant number: 14/04477-9
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: August 01, 2014
• End date: November 30, 2017
• Field of knowledge: Biological Sciences - Biophysics - Radiology and Photobiology
• Agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal Investigator: Vadim Viviani
• Grantee: Gabriele Verônica de Mello Gabriel
• Host Institution: Centro de Ciências e Tecnologias para a Sustentabilidade (CCTS). Universidade Federal de São Carlos (UFSCAR). Sorocaba , SP, Brazil
• Associated scholarship(s): 16/15946-5 - Expression of Macrolampis sp2 firefly luciferase in mammalian cells for intracelular pH-biosnsing, BE.EP.DR 15/22603-4 - Humanization and expression of Macrolampis sp2 firefly luciferase for intracelular pH-biosensing, BE.EP.DR

Abstract

Among various applications of bioluminescence, its use in biosensors is increasing. The biosensors have several advantages over conventional detection techniques, such as low production cost, high sensitivity and practical use. A bioluminescent biosensor can be enzymatic, which employ immobilized luciferases enzymes, or cellular, which are constructed by genetic transformation of cells with luciferase reporter gene harboring expression vectors. These types of biosensors can be used to indicate the presence of toxic compounds, such as metals, or changes in cellular metabolism by light emission. Specific biosensors for intracellular pH monitoring are extremely important to analyze biological processes such as apoptosis and oncogenesis. Moreover, specific biosensors for the detection of metals in environmental samples are also on demand. Firefly luciferases have the bioluminescence spectrum which is sensitive to pH and the presence of metals. Nevertheless, this property has never been used for the purpose of pH sensing or detection of metals. In 2006, the supervisor of this project filed a patent claiming the use of Macrolampis sp2 luciferase and its pH sensitivity for pH and metals biosensing, however until now no specific biosensor has been developed in this context. Therefore, the main goals of this project are: (I) to investigate the suitability of Macrolampis sp2 firefly luciferase and other pH-sensitive luciferases cloned by the research group as spectral ratiometric intracellular pH biosensors, (II) to develop, a ratiometric biosensor for specific metals by engineering the pH-sensing moiety of Macrolampis sp2 luciferase, and (III) to apply these ratiometric biosensors in mammalian cells to investigate their physiology in real time. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

DE MELLO GABRIEL, GABRIELE VERONICA; YASUNO, RIE; MITANI, YASUO; OHMIYA, YOSHIHIRO; VIVIANI, VADIM RAVARA. Novel application of Macrolampis sp2 firefly luciferase for intracellular pH-biosensing in mammalian cells. PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, v. 18, n. 5, p. 1212-1217, MAY 1 2019. (10/05426-8, 14/04477-9, 16/15946-5, 11/23961-0, 15/22603-4)

GABRIEL, GABRIELE V. M.; VIVIANI, VADIM R.. Novel application of pH-sensitive firefly luciferases as dual reporter genes for simultaneous ratiometric analysis of intracellular pH and gene expression/location. PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, v. 13, n. 12, p. 1661-1670, 2014. (11/23961-0, 14/04477-9)

GABRIEL, GABRIELE V. M.; VIVIANI, VADIM R.. Engineering the metal sensitive sites in Macrolampis sp2 firefly luciferase and use as a novel bioluminescent ratiometric biosensor for heavy metals. ANALYTICAL AND BIOANALYTICAL CHEMISTRY, v. 408, n. 30, p. 8881-8893, DEC 2016. (11/23961-0, 14/04477-9, 13/09594-0)