Applied Biochemistry and Biotechnology

, Volume 172, Issue 4, pp 2175–2187 | Cite as

Development of a Biosensor for Copper Detection in Aqueous Solutions Using an Anemonia sulcata Recombinant GFP

  • Tiziana Masullo
  • Riccardo Puccio
  • Mariarosaria Di Pierro
  • Marcello Tagliavia
  • Paolo Censi
  • Valeria Vetri
  • Valeria Militello
  • Angela Cuttitta
  • Paolo Colombo
Article
First Online:
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Accepted:

Abstract

Fluorescent proteins from marine organisms represent potential candidates for biosensor development. In this paper, we described the isolation of a native green fluorescent protein from Anemonia sulcata and the cloning and purification of its equivalent as a recombinant protein in Escherichia coli. Furthermore, the spectroscopic behaviours of the native and recombinant GFPs were investigated as a function of Cu2+, Cd2+, Pb2+ and Ni2+ concentration. Our results suggest the high selectivity of both proteins at copper than the other metals and, for the recombinant protein, a great sensitivity at a very low concentration (0.1–1 μM). Moreover, starting from these data, using the combination of molecular biology techniques and optical setup, we developed a device for the detection of Cu2+ in water solutions. The quenching effect detected with the device showed that the relative attenuation of the signal (0.46 ± 0.02 AU) was slightly larger than the data measured by fluorescence spectra (0.65 ± 0.03 AU). The good sensitivity in the span of two orders of the magnitude of Cu2+ concentration, the fact that the instrument is made up of low-cost and sturdy parts and the selective quenching of rAsGFP to copper ions make this setup suited as a low cost, on-the-field, copper ion-specific biosensor.

Keywords

Green fluorescent protein Recombinant protein Divalent metal ions Copper Quenching Detector 
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Notes

Acknowledgments

We thank Dr. Salvatore Mazzola for critical reading of the manuscript and Dr. Aldo Nicosia and Dr. Monica Salamone for helpful discussion. This work was supported by PO FERS 2007/2013 Linea di intervento 4.1.1.2.

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Tiziana Masullo
    • 1
  • Riccardo Puccio
    • 2
  • Mariarosaria Di Pierro
    • 3
  • Marcello Tagliavia
    • 2
  • Paolo Censi
    • 1
    • 4
  • Valeria Vetri
    • 5
    • 6
  • Valeria Militello
    • 5
    • 6
  • Angela Cuttitta
    • 1
  • Paolo Colombo
    • 7
  1. 1.Istituto per l’Ambiente Marino Costiero, UOS Capo Granitola—Consiglio Nazionale delle RicercheTrapaniItaly
  2. 2.BioNat Italia S.r.l.PalermoItaly
  3. 3.Biodiagene S.r.l.PalermoItaly
  4. 4.Dipartimento di Scienze della Terra e del Mare DiSTeMUniversità di PalermoPalermoItaly
  5. 5.Dipartimento di Fisica e ChimicaUniversità di PalermoPalermoItaly
  6. 6.Istituto di Biofisica—Consiglio Nazionale delle RicerchePalermoItaly
  7. 7.Istituto di Biomedicina e di Immunologia Molecolare—Consiglio Nazionale delle RicerchePalermoItaly

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