Fluorescent ProteinBased Optical Biosensor for Co

Authors: Chartchalerm IsarankuraNaAyudhya Tanawut Tantimongcolwat HansJoachim Galla Virapong Prachayasittikul

Publish Date: 2009/08/01

Volume: 134, Issue: 3, Pages: 352-363

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Abstract

In the present study spectroscopic determinations of copper ions using chimeric metalbinding green fluorescent protein His6GFP as an active indicator have been explored Supplementation of copper ions to the GFP solution led to a remarkable decrease of fluorescent intensity corresponding to metal concentrations For circumstances rapid declining of fluorescence up to 60 was detected in the presence of 500 μM copper This is in contrast to those observed in the case of zinc and calcium ions in which approximately 10–20 of fluorescence was affected Recovery of its original fluorescence up to 80 was mediated by the addition of ethylenediamine tetraacetic acid More importantly in the presence of metal ions the emission wavelength maximum remains unchanged while reduction of the optical density of the absorption spectrum has been observed This indicates that the chromophore’s ground state was possibly affected by the static quenching process Results from circular dichroism measurements revealed that the overall patterns of circular dichroism spectra after exposure to copper ions were not significantly different from that of the control where the majority of sharp positive band around 195–196 nm in combination with a broad negative deflection around 215–216 nm was obtained Taken together it can be presumed that copper ions exerted their static quenching on the fluorescence rather than structural or conformational alteration However notification has to be made that some peptide rearrangements may also occur in the presence of metal ions Further studies were conducted to investigate the feasibility of using the His6GFP as a sensing unit for copper ions The His6GFP was encapsulated in Solgel and immobilized onto the optical fiber connected with a fluorescence detecting device The Solgel was doped into the metal solution where the quenching of fluorescence could be monitored in real time The sensing unit provided a high sensitivity of detection in the range of 05 μM to 50 mM with high selectivity for copper ions All these findings open up a high potential to apply the fluorescent proteinbased bioanalytical tool for copper determination in the futureThis project was supported in part by the Young Scholars Research Fellowship from The Thailand Research Fund to TT grant no MRG5080158 the Deutsche Forschungsgemeinschaft DFG and the Bundesministerium fur wirtschaftliche Zusammenarbeit und Entwicklung Federal Ministry for Economic Cooperation and Development BMZ grant noGA233/19–12 and the annual budget grant of Mahidol University BE 25512555 The authors would like to thank Prof Dr HansUlrich Humpf Institut für Lebensmittelchemie Westfälische Wilhelms Universität for his assistance on the operation of CD measurements

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