Arsenate reductase from Thermus thermophilus conjugated to polyethylene glycol-stabilized gold nanospheres allow trace sensing and speciation of arsenic ions

Abstract

Water sources pollution by arsenic ions is a serious environmental problem all around the world. Arsenate reductase enzyme (TtArsC) from Thermus thermophilus extremophile bacterium, naturally binds arsenic ions, As(V) and As (III), in aqueous solutions. In this research, TtArsC enzyme adsorption onto hybrid polyethylene glycol-stabilized gold nanoparticles (AuNPs) was studied at different pH values as an innovative nanobiosystem for metal concentration monitoring. Characterizations were performed by UV/Vis and circular dichroism spectroscopies, TEM images and in terms of surface charge changes. The molecular interaction between arsenic ions and the TtArsC-AuNPs nanobiosystem was also monitored at all pH values considered by UV/Vis spectroscopy. Tests performed revealed high sensitivities and limits of detection equal to 10 ± 3 M^-12 and 7.7 ± 0.3 M^-12 for As(III) and As(V), respectively.

Keywords: arsenic pollution; enzyme; gold nanoparticles.

Figure 1.

Z-potential evaluation highlights how surface charge of TtArsC changes when bioconjugated to AuNPs (TtArsC-AuNPs) (a); CD measurements reveals slight TtArsC enzyme rearrangement onto AuNPs surface (b). (Online version in colour.)

Figure 2.

Z-potential evaluation highlights that surface charge of TtArsC-AuNPs at pH values from 5 to 9 does not significantly changes (a), even though CD measurements demonstrate TtArsC-AuNPs enzyme undergoes rearrangement of secondary structure (b). Different colour solutions are visible at naked-eye as function of pH values (c). (Online version in colour.)

Figure 3.

LSP shift as function of As(III) ions increasing concentration in solution (a); LSP change trend as function of As(III) ions increasing concentration in solution, normalized to LSP values in the absence of As(III) (b). In the inset, signals generated by TtArsC-AuNPs on exposure to small concentrations of As(III) ions. (Online version in colour.)

Figure 4.

LSP shift as function of As(V) ions increasing concentration in solution (a); LSP change trend as function of As(V) ions increasing concentration in solution, normalized to LSP value in the absence of As(V) (b). In the inset, signals generated by TtArsC-AuNPs on exposure to small concentrations of As(V). (Online version in colour.)

Figure 5.

Digital images of TtArsC-AuNPs mixtures in eppendorfs at low concentration of arsenic ions which clearly demonstrate naked-eye assay in monitoring the presence of this heavy metal in solution. (Online version in colour.)

Figure 6.

TEM images of TtArsC-AuNPs nanocomplex after bioconjugation of enzyme and PEG-stabilized gold nanoparticles (a); image of a cluster of TtArsC-AuNPs on exposure to As(III) ions at 8.5 µM (b); image of a cluster of TtArsC-AuNPs on exposure to As(V) at 8.5 µM (c).

Figure 7.

Trend of surface charge (Z-potential values) changes after 24 h as function of different pH values in the absence and on exposure to arsenic ions (a); naked-eye evidence of TtArsC-AuNPs solutions colour change at different pH values in the presence of As(III) (b) and As(V) (c). (Online version in colour.)

Figure 8.

TtArsC-AuNPs LSP signal changes at different pH values (ON stands for overnight incubation) in the absence and on exposure to arsenic ions As(III) and As(V) at 8.5 µM (from 5 (a) to 9 (e)); LSP values as function of pH values in the absence and on exposure to arsenic ions normalized on LSP values in the absence of arsenic ions (f). (Online version in colour.)