Towards single cell ICP-MS normalized quantitative experiments using certified selenized yeast.

In the search for a normalized procedure to replicate and compare single cell-inductively coupled plasma-mass spectrometry (SC-ICP-MS) experiments, SELM-1, a certified reference material containing selenium enriched yeast cells has been used. Selenium concentrations (both, intra- and extracellular) have been measured using either sequential or simultaneous procedures. Regarding quantitative results, the sequential procedure involving cell washing followed by freeze drying of the washed material and intracellular Se quantification using SC-ICP-MS provided best results. In this case, intracellular Se accounted for 1304 ± 48 mg kg-1 (corresponding to 64% of the certified Se content). The average mass of Se per yeast cell was 41.6 fg Se with a dispersion of 1.6-279 fg Se/cell. In the isolated extracellular Se fraction, the Se concentration accounted for 412 ± 48 mg kg-1 (about 21% of the total Se). Thus, the sequential procedure provided a total Se recovery of about 85% with respect to the certified value. The direct dilution and simultaneous measurement of intra- and extracellular Se by SC-ICP-MS provided results of 1024 ± 42 mg kg-1 for intracellular and 316 ± 30 mg kg-1 for extracellular Se representing a total recovery of about 66%. In both cases, an initial thorough characterization of the cell density per solid weighed material was conducted by flow cytometry and the cell integrity ensured using confocal microscopy. These results clearly demonstrated that with appropriate sample preparation, SC-ICP-MS is a unique tool, which is capable of providing quantitative information about intracellular and extracellular Se. In addition, SELM-1 seems the ideal tool to enable data normalization at the single cell level to replicate, benchmark, and improve new SC-ICP-MS studies by using the same material for data validation.

Addressing the presence of biogenic selenium nanoparticles in yeast cells: analytical strategies based on ICP-TQ-MS.

Several organisms have demonstrated the ability of synthesising biogenic selenium-containing nanoparticles. Such particles from biological sources have attracted great attention due to several proven activities as antioxidants or antimicrobial agents. However, little is known in terms of size (distribution), shapes, chemical composition and number/amount/concentration of these particles. Therefore, in this work, we proposed the use of complementary analytical strategies that enabled the detection and characterization of selenium-containing nanoparticles in selenized yeast (Saccharomyces cerevisiae). The first strategy to address the intracellular presence of Se within yeast cells, involves the use of single cell ICP-TQ-MS (inductively coupled plasma-mass spectrometry). For this aim, selenium and phosphorous (as constitutive element) were measured as oxides (80Se16O+ and 31P16O+, resp.) in the triple-quadrupole mode. Then, a simple and fast cell lysis by mechanical disruption is conducted (approx. 30 min) in order to prove the presence of selenium-containing nanoparticles (SeNPs). The lysate is analysed by single particle ICP-TQ-MS and, complementarily, by liquid chromatography coupled to ICP-TQ-MS to cover a wider range of particle sizes. One of the samples revealed the presence of dispersed SeNPs with sizes between a few nm and up to 250 nm also confirmed by transmission electron microscopy (TEM) in the form of elemental selenium. The analysis of the certified reference material SELM-1 showed the presence of spherical SeNPs of 4 to 7 nm diameter. These biogenic particles, at least partially, were made of elemental selenium as well. The whole study reveals the excellent capabilities of "single" event ICP-MS methodologies in combination with HPLC-based strategies for a complete characterization of nanoparticulated material in biological samples.

Quantitative Evaluation of Cisplatin Uptake in Sensitive and Resistant Individual Cells by Single-Cell ICP-MS (SC-ICP-MS).

One of the main limitations to the Pt-therapy in cancer is the development of associated drug resistance that can be associated with a significant reduction of the intracellular platinum concentration. Thus, intracellular Pt concentration could be considered as a biomarker of cisplatin resistance. In this work, an alternative method to address intracellular Pt concentration in individual cells is explored to permit the evaluation of different cell models and alternative therapies in a relatively fast way. For this aim, total Pt analysis in single cells has been implemented using a total consumption nebulizer coupled to inductively coupled plasma mass spectrometric detection (ICP-MS). The efficiency of the proposed device has been evaluated in combination with flow cytometry and turned out to be around 25% (cells entering the ICP-MS from the cells in suspension). Quantitative uptake studies of a nontoxic Tb-containing compound by individual cells were conducted and the results compared to those obtained by bulk analysis of the same cells. Both sets of data were statistically comparable. Thus, final application of the developed methodology to the comparative uptake of Pt-species in cisplatin resistant and sensitive cell lines (A2780cis and A2780) was conducted. The results obtained revealed the potential of this analytical strategy to differentiate between different cell lines of different sensitivity to the drug which might be of high medical interest.