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

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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.

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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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Authors and Affiliations

  1. Istituto per l’Ambiente Marino Costiero, UOS Capo Granitola—Consiglio Nazionale delle Ricerche, Trapani, Italy

    Tiziana Masullo, Paolo Censi & Angela Cuttitta

  2. BioNat Italia S.r.l., Palermo, Italy

    Riccardo Puccio & Marcello Tagliavia

  3. Biodiagene S.r.l., Palermo, Italy

    Mariarosaria Di Pierro

  4. Dipartimento di Scienze della Terra e del Mare DiSTeM, Università di Palermo, Palermo, Italy

    Paolo Censi

  5. Dipartimento di Fisica e Chimica, Università di Palermo, Palermo, Italy

    Valeria Vetri & Valeria Militello

  6. Istituto di Biofisica—Consiglio Nazionale delle Ricerche, Palermo, Italy

    Valeria Vetri & Valeria Militello

  7. Istituto di Biomedicina e di Immunologia Molecolare—Consiglio Nazionale delle Ricerche, Via Ugo La Malfa 153, 90146, Palermo, Italy

    Paolo Colombo

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  1. Tiziana Masullo
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  2. Riccardo Puccio
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  8. Angela Cuttitta
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  9. Paolo Colombo
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Correspondence to Paolo Colombo.

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Masullo, T., Puccio, R., Di Pierro, M. et al. Development of a Biosensor for Copper Detection in Aqueous Solutions Using an Anemonia sulcata Recombinant GFP. Appl Biochem Biotechnol 172, 2175–2187 (2014). https://doi.org/10.1007/s12010-013-0669-1

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  • DOI: https://doi.org/10.1007/s12010-013-0669-1

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