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

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 Cu 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 Appl Biochem Biotechnol (2014) techniques and optical setup, we developed a device for the detection of Cu 2+ 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 Cu 2+ 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-thefield, copper ion-specific biosensor.

Crystal structures of Dronpa complexed with quenchable metal ions provide insight into metal biosensor development

“…Particularly, the amino acid sequence of His210 and His212 ( i and i + 2) on the β12‐strand of Dronpa was identical to that previously reported in robust transition metal binding in a β‐sheet mode . Many FPs have shown Cu 2+ ‐induced fluorescence quenching , but the configuration of the Cu 2+ binding was still unknown. Thus, determination of the Cu 2+ ‐binding site in Dronpa could be useful to understand other Cu 2+ sensitive FPs.…”

“…One of the interesting properties of FPs is fluorescence quenching by specific divalent metal ions . Many fluorescent proteins have been used for biosensing applications to detect transition metal ions . For instance, DsRed from a reef coral has been reported as a copper sensor with highly selective, reversible and sensitive detection capacities for both Cu + and Cu 2+ , and a detection limit in the nanomolar range .…”

Chromophorylation of a Novel Cyanobacteriochrome GAF Domain from Spirulina and Its Response to Copper Ions

“…The fluorescence of SPI1085g2-PEB can be selectively and instantaneously quenched by Cu 2+ in a concentration-dependent manner and recovered almost to the initial fluorescence with the addition of EDTA. SPI1085g2-PEB provided a much faster response time compared to other fluorescent proteins, such as His6GFP (chimeric green fluorescent protein harboring hexahistidine) [ 28 ], rAsGFP [ 29 ], and iLOV (variant derived through engineering of the light, oxygen or voltage sensing domain) [ 30 ], which suggests the potential of SPI1085g2-PEB as a biosensor for rapid detection of copper ions. In this case, the observed fluorescence changes may be due to the interaction between Cu 2+ and metal-binding sites, which presumably cause static quenching, as well as that of His6GFP, which bring copper close to the chromophore of the proteins to form a nonfluorescent ground state complex.…”

Screen-Printed Voltammetric Biosensors for the Determination of Copper in Wine

“…From this point of view, the use of screen-printed electrodes not only solves the problem of cost efficiency, but also satisfies the portability requirement, contributing to the progress towards decentralized analysis. There are numerous studies on the detection of copper ions with biosensors by the specified means, but most of them are developed around the analysis of these ions in water or foods [4,5,6,7,8]. Only a few studies can be applied in the determination of the copper content of wine, despite the fact that the presence of this element has a great impact on the quality and safety of this product [2,9].…”