Analytical and Bioanalytical Chemistry

, Volume 408, Issue 30, pp 8881–8893 | Cite as

Engineering the metal sensitive sites in Macrolampis sp2 firefly luciferase and use as a novel bioluminescent ratiometric biosensor for heavy metals

  • Gabriele V. M. Gabriel
  • Vadim R. VivianiEmail author
Research Paper
Part of the following topical collections:
  1. Highlights of Analytical Chemical Luminescence


Most luminescent biosensors for heavy metals are fluorescent and rely on intensity measurements, whereas a few are ratiometric and rely on spectral changes. Bioluminescent biosensors for heavy metals are less common. Firefly luciferases have been coupled to responsive promoters for mercury and arsenium, and used as light on biosensors. Firefly luciferase bioluminescence spectrum is naturally sensitive to heavy metal cations such as zinc and mercury and to pH. Although pH sensitivity of firefly luciferases was shown to be useful for ratiometric estimation of intracellular pH, its potential use for ratiometric estimation of heavy metals was never considered. Using the yellow-emitting Macrolampis sp2 firefly luciferase and site-directed mutagenesis, we show that the residues H310 and E354 constitute two critical sites for metal sensitivity that can be engineered to increase sensitivity to zinc, nickel, and mercury. A linear relationship between cation concentration and the ratio of bioluminescence intensities at 550 and 610 nm allowed, for the first time, the ratiometric estimation of heavy metals concentrations down to 0.10 mM, demonstrating the potential applicability of firefly luciferases as enzymatic and intracellular ratiometric metal biosensors.


Metal binding sites Luciferases pH sensitivity Reporter gene Metal biosensor Ratiometric curve 



Macrolampis sp2 firefly luciferase with substitution of histidine to alanine on residue 310


Macrolampis sp2 firefly luciferase with substitution of histidine to cysteine on residue 310


Macrolampis sp2 firefly luciferase with substitution of histidine to cysteine on residue 310 and asparagine to cysteine on residue 354.


Macrolampis sp2 firefly luciferase with substitution of asparagine to cysteine on residue 354


Macrolampis sp2 firefly luciferase with substitution of asparagine to glutamic acid on residue 354


Macrolampis sp2 firefly luciferase with substitution of asparagine to histidine on residue 354


Macrolampis sp2 firefly luciferase



We thank São Paulo Research Foundation (FAPESP) (grant #2011/23961-0; #2013/09594-0 and #2014/04477-9) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) 477616/2012-0 for financial support.

Compliance with ethical standards

Conflict of interest

A patent (Brazilian patent PI0604475-1 A2, 2006) using Macrolampis sp2 firefly luciferase spectral sensitivity to detect metal has been applied for. No other conflict of interest is found.

Supplementary material

216_2016_11_MOESM1_ESM.pdf (1.4 mb)
ESM 1 (PDF 1.35 mb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Gabriele V. M. Gabriel
    • 1
    • 2
  • Vadim R. Viviani
    • 1
    • 2
    • 3
    Email author
  1. 1.Laboratory of Biochemistry and Biotechnology of Bioluminescence, Department of Physics, Chemistry and MathematicsFederal University of São Carlos (UFSCar), Campus of SorocabaSorocabaBrazil
  2. 2.Graduate School of Evolutive Genetics and Molecular BiologyFederal University of São Carlos (UFSCar)São CarlosBrazil
  3. 3.Graduate School of Biotechnology and Environmental MonitoringFederal University of São Carlos (UFSCar), Campus of SorocabaSorocabaBrazil

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