Evaluation of the Transformation of Selenite and Selenium Nanoparticles to Seleno-Amino Acids Produced by Escherichia Coli and Staphylococcus Aureus by Using Liquid Chromatography -Inductively Coupled Plasma Mass Spectrometry and Single-Particle- Inductively Coupled Plasma Mass Spectrometry and Different Sample Treatments

Due to the scarce knowledge about the impact of selenium nanoparticles (SeNPs) on bacterial populations, the main objective of this work was focused on evaluating the transformations of SeNPs and selenite in Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). For this purpose, an analytical methodology based on bacteria cell wall disruption, carbamidomethylation, enzymatic hydrolysis and high-performance liquid chromatography couple to inductively coupled plasma mass spectrometry (HPLC-ICP-MS) and HPLC- electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) measurements was developed, the latter for unambiguous identification of selenium species. Once bacteria were cultured in the presence of chitosan modified SeNPs (Ch-SeNPs) and selenite at 0, 1 and 2 mg L-1 Se for 24 h, an enzymatic disruption of the bacterial cell wall using lysozyme followed by enzymatic hydrolysis with protease was applied. The use of lysozyme to extract selenium species provided a better efficiency in the total selenium content (higher than 96%), compared to a mechanical disruption of the bacterial cell wall. Analysis of the extracts by anionic exchange HPLC-ICP-MS showed a strong influence of incubation time with protease (24, 48 and 72 h) on selenium chromatographic profile. The results showed that selenocysteine (SeCys) was the only Se species identified in both bacteria representing an 80% of total selenium accumulated. The confirmation of the identity of this Se species was carried out after performing a carbamidomethylation process, prior to enzymatic hydrolysis, and analyzing the extract by reversed phase HPLCESI-MS/MS. These analyses confirmed the presence of SeCys, and no relevant differences were found between the metabolic pathway of both forms of selenium. Moreover, the growth of both bacterial species in the presence of selenite resulted in the formation of biogenic SeNPs. Characterization by TEM before and after their separation from the culture medium showed spherical and monodispersed nanoparticles with an average size (155 & PLUSMN; 19) nm and (172 & PLUSMN; 20) nm for E. coli and S. aureus, respectively. Analysis by spICP-MS showed no significant differences in size with respect to TEM after considering the ionization efficiency of Se.