Biomimetic Synthesis of Selenium Nanospheres by Bacterial Strain JS-11 and Its Role as a Biosensor for Nanotoxicity Assessment: A Novel Se-Bioassay

<div><p>Selenium nanoparticles (Se-NPs) were synthesized by green technology using the bacterial isolate <i>Pseudomonas aeruginosa</i> strain JS-11. The bacteria exhibited significant tolerance to selenite (SeO₃²⁻) up to 100 mM concentration with an EC₅₀ value of 140 mM. The spent medium (culture supernatant) contains the potential of reducing soluble and colorless SeO₃²⁻ to insoluble red elemental selenium (Se⁰) at 37°C. Characterization of red Se° product by use of UV-Vis spectroscopy, X-ray diffraction (XRD), atomic force microscopy (AFM) and transmission electron microscopy (TEM) with energy dispersive X-ray spectrum (EDX) analysis revealed the presence of stable, predominantly monodispersed and spherical selenium nanoparticles (Se-NPs) of an average size of 21 nm. Most likely, the metabolite phenazine-1-carboxylic acid (PCA) released by strain JS-11 in culture supernatant along with the known redox agents like NADH and NADH dependent reductases are responsible for biomimetic reduction of SeO₃²⁻ to Se° nanospheres. Based on the bioreduction of a colorless solution of SeO₃²⁻ to elemental red Se⁰, a high throughput colorimetric bioassay (Se-Assay) was developed for parallel detection and quantification of nanoparticles (NPs) cytotoxicity in a 96 well format. Thus, it has been concluded that the reducing power of the culture supernatant of strain JS-11 could be effectively exploited for developing a simple and environmental friendly method of Se-NPs synthesis. The results elucidated that the red colored Se° nanospheres may serve as a biosensor for nanotoxicity assessment, contemplating the inhibition of SeO₃²⁻ bioreduction process in NPs treated bacterial cell culture supernatant, as a toxicity end point.</p> </div

XRD pattern of the bacterial Se-NPs.

<p> The characteristic strong diffraction peak located at 31.64° is ascribed to the (101) facets of the face-centred cubic elemental Se° structure.</p

Selenite tolerance by <i>Pseudomonas aeruginosa</i> strain JS-11.

<p>Selenite tolerance by <i>Pseudomonas aeruginosa</i> strain JS-11.</p

FTIR analysis of PCA.

<p>The spectra depict the changes in the peaks of PCA alone (red) and after treatment with 2 mM Na₂SeO₃^2<b>−</b> solution (blue).</p

HPLC analysis of PCA produced by the strain JS-11.

<p>HPLC profile indicating the PCA peak at retention time of 9.6 min. Inset shows the (A): bacterial culture supernatant, (B): Benzene extract of PCA, (C): PCA after extraction and (D) PCA crystals.</p

UV-Visible absorption spectra of extracellularly synthesized Se-NPs.

<p>The typical surface plasmon resonance (SPR) band is shown at 520 nm. The labels A–D represent 2, 24, 48 and 72 h of incubation, respectively. Inset depicts the change in color of culture supernatant from pale yellow to red after 24 h of incubation with 2 mM Na₂SeO₃^2<b>−</b> solution.</p

NADH fluorescence of bacterial supernatant alone and after treatment with NPs.

<p>The arrow represents the fluorescence quench of spectra A–F, where A is untreated control supernatant, and spectra B to F represent the supernatant treated with TiO₂-NPs, ZnFe₂O₄-NPs, CdS-NPs, Ag-NPs (100 µgml<b>⁻</b>¹) and EMS (2 mM) in a total volume of 3 ml, respectively.</p

Microscopic analysis of Se-NPs produced by strain JS-11.

<p>Panel (A) shows the representative transmission electron micrograph recorded from a drop-coated film of the aqueous solution of Se-NPs; Panel (B) represents the energy dispersive X-ray spectrum of Se-NPs; (C) represents the average hydrodynamic size and zeta potential of Se-NPs; and (D) represents the 3D topography of Se-NPs in top view (scan size is 5×5 µm) by atomic force microscopic analysis.</p