Fluorescent Protein-Based Optical Biosensor for Copper Ion Quantitation

Abstract

In the present study, spectroscopic determinations of copper ions using chimeric metal-binding 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 microM 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 Sol-gel and immobilized onto the optical fiber connected with a fluorescence detecting device. The Sol-gel 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 0.5 microM to 50 mM with high selectivity for copper ions. All these findings open up a high potential to apply the fluorescent protein-based bioanalytical tool for copper determination in the future.

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

“…It is noteworthy to mention that our AsGFP is produced as a His-tag protein, so it can be suggested that the presence of additional histidine residues can enhance the quenching effect as shown for other GFPs. Many authors have confirmed a selective bond of copper with mutated GFP, in which some residues are changed to histidines [8,10,13], and have emphasized how the location and distance of the imidazole ring need to be very close to the GFP fluorophore in order to effectively quench the fluorescence [12].…”

“…Fluorescence quenching has been observed by Hg 2+ [5] and Cu 2+ on an engineered chimeric GFP harbouring hexahistidine [8]. Moreover, the introduction of metalbinding sites onto the GFP surface places metals in close proximity to chromophore resulted in fluorescence quenching probably induced by energy transfer, at low concentrations (micromolar) of copper ions [10].…”

“…For instance, fluorescent proteins (FPs) from different marine organisms may become potential candidates for sensor development. In fact, FPs, such as red and green (GFP) proteins, were used to evaluate the effects of heavy and essential metals on fluorescence intensity [5][6][7][8][9][10][11][12].…”

Fluorescence of a Histidine-Modified Enhanced Green Fluorescent Protein (EGFP) Effectively Quenched by Copper(II) Ions. Part II. Molecular Determinants

“…It was found that the genetically encoded fluorescent sensors based on green fluorescent protein can be applied to analyze the availability of copper in intracellular processes. Many of these applications require reporters with specific physicochemical properties which lead to continuous apparition of genetically modified fluorescent protein variants [8][9][10][11][12][13][14].…”

An Engineered Palette of Metal Ion Quenchable Fluorescent Proteins

“…Thus, metal-induced changes in fluorescence can be used to report the presence of specific metals in a solution or cell. The change in fluorescence induced by metals can occur either by static quenching [12], energy transfer between a colored metal ion and the chromophore [11], or by perturbations to the protein’s structure [10]. In some designs, cross-bridging metal sites have been added to a linker that connects two differently-colored FPs [13]–[15].…”