ABSTRACT
Current sample preparation strategies for nanomaterials (NMs) analysis in soils by means single particle inductively coupled plasma mass spectrometry have significant constrains in terms of accuracy, sample throughput and applicability (i.e., type of NMs and soils). In this work, strengths and weakness of microwave assisted extraction (MAE) for NMs characterization in soils were systematically investigated. To this end, different extractants were tested (ultrapure water; NaOH, NH4OH, sodium citrate and tetrasodium pyrophosphate) and MAE operating conditions were optimized by means of design of experiments. Next, the developed method was applied to different type of metallic(oid) nanoparticles (Se-, Ag-, Pt- and AuNPs) and soils (alkaline, acid, sandy, clayey, SL36, loam ERMCC141; sludge amended ERM483). Results show that Pt- and AuNPs are preserved and quantitatively extracted from soils in 6 min (12 cycles of 30 s each) inside an 800 W oven by using 20 mL of 0.1 M NaOH solution. This methodology is applicable to soils showing a wide range of physicochemical properties except for clay rich samples. If clay soil fraction is significant (>15%), NMs are efficiently retained in the soil thus giving rise to poor recoveries (<10%). The analysis of labile NMs such as Se- and AgNPs is not feasible by means this approach since extraction conditions favors dissolution. Finally, when compared to current extraction methodologies (e.g., ultrasound, cloud point extraction, etc.), MAE affords better or equivalent accuracies and precision as well as higher sample throughput due to treatment speed and the possibility to work with several samples simultaneously.
ABSTRACT
One of the greatest challenges of implementing fast molecular detection methods as part of Legionella surveillance systems is to limit detection to live cells. In this work, a protocol for sample treatment with propidium monoazide (PMA) in combination with quantitative PCR (qPCR) has been optimized and validated for L. pneumophila as an alternative of the currently used time-consuming culture method. Results from PMA-qPCR were compared with culture isolation and traditional qPCR. Under the conditions used, sample treatment with 50 µM PMA followed by 5 min of light exposure were assumed optimal resulting in an average reduction of 4.45 log units of the qPCR signal from heat-killed cells. When applied to environmental samples (including water from cooling water towers, hospitals, spas, hot water systems in hotels, and tap water), different degrees of correlations between the three methods were obtained which might be explained by different matrix properties, but also varying degrees of non-culturable cells. It was furthermore shown that PMA displayed substantially lower cytotoxicity with Legionella than the alternative dye ethidium monoazide (EMA) when exposing live cells to the dye followed by plate counting. This result confirmed the findings with other species that PMA is less membrane-permeant and more selective for the intact cells. In conclusion, PMA-qPCR is a promising technique for limiting detection to intact cells and makes Legionella surveillance data substantially more relevant in comparison with qPCR alone. For future research it would be desirable to increase the method's capacity to exclude signals from dead cells in difficult matrices or samples containing high numbers of dead cells.
