Measurement of the copper concentration in drinking water based on changes of the fluorescence lifetime of the green fluorescent protein

摘要

Copper is a heavy metal, which is used in heat and electrical conductors and in a multitude of alloys in the technical context. Moreover, it is a trace element that is essential for the life of organisms but can cause toxic effects in elevated concentrations. Maximum limits in water and for beverages exist. Beyond that there is a need for the control of copper concentrations in the fields of sewage and agriculture. Hence, competitive measurement systems that allow for the fast, user-friendly and reliable detection are presumed to have a large potential market. One prominent class of naturally occurring fluorophores is the Green Fluorescent Protein (GFP). GFP originally stems from the jellyfish Aequorea Victoria and has found its way in various applications e.g. as biosensors in basic research and for monitoring gene expression. Exploiting GFP in plant cells allowed for the visualization of the copper uptake by changes in the GFP fluorescence. In principle changes in the fluorescence intensity or in the fluorescence lifetime can be utilized to determine copper concentrations. However, lifetime changes have the advantage of omitting calibration measurements and therefore make this method ideally suited for sensing purposes. Here the decrease of the fluorescence lifetime of GFP by Foerster resonance energy transfer (FRET) is used to measure the copper ion concentration in drinking water. Therefore a system is developed that is based on a GFP sample in a predefined concentration. The GFP mutant can be excited with blue light. For binding of copper ions a His-tag is included in the GFP. After measuring the fluorescence lifetime of the pure GFP the copper determination of the sample is performed by lifetime measurement. Therefore the lifetime can be assigned to the copper concentration of the GFP-doped drinking water sample. In summary a method for the quantification of copper ions based on changes of the fluorescence lifetime of GFP is developed and the measurement of the copper concentration in water samples is performed.