Article Cite This: Environ. Sci. Technol. XXXX, XXX, XXX−XXX
pubs.acs.org/est
Quantification of Au Nanoparticle Biouptake and Distribution to Freshwater Algae Using Single Cell − ICP-MS R. C. Merrifield,*,†,§ C. Stephan,‡ and J. R. Lead*,† †
Centre for Environmental Nanoscience and Risk, University South Carolina, Columbia, South Carolina 29208, United States PerkinElmer, Woodbridge, Ontario L4L 8H1, Canada
‡
S Supporting Information *
ABSTRACT: Quantifying metal and nanoparticle (NP) biouptake and distribution on an individual cellular basis has previously been impossible, given available techniques which provide qualitative data that are laborious to acquire and prone to artifacts. Quantifying metal and metal NP uptake and loss processes in environmental organisms will lead to mechanistic understanding of biouptake and improved understanding of potential hazards and risks of metals and NPs. In this work, we present a new technique, single cell inductively coupled plasma mass spectrometry (SC-ICP-MS), which allows quantification of metal concentrations on an individual cell basis down to the attogram (ag) per cell level. We present data validating the novel method, along with the mass of metal per cell. Finally, we use SC-ICP-MS, with ancillary cell counting methods, to quantify the biouptake and strong sorption and distribution of both dissolved Au and Au NPs in a freshwater alga (Cyptomonas ovate). The data suggests differences between dissolved and NP uptake and loss. In the case of NPs, there was a dose and time dependent uptake, but individual cellular variations; at the highest realistic exposure conditions used in this study up to 40−50% of cells contained NPs, while 50−60% of cells did not.
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INTRODUCTION
often overlooked. Typically a nominal added mass concentration is taken as the exposure, despite known issues with poor characterization,15 sorptive loss, and transformations, such as aggregation, dissolution, and surface alterations.16−18 Vital information is lost about the concentration and form of the NP to which the cells are exposed, and whether this alters during the exposure period. In addition, nominal dose is measured as the averaged mass concentration of the entire cell population, using the assumption that all cells uptake and accumulate metals and NPs equally, which is unlikely. Biological differences, along with the dynamic nature of NP transformations, suggest the need for better measurement of exposure and dose.19 Currently, there is no analytical methodology available to interrogate uptake and internalized cellular NP concentrations on a single cell basis.20−22 At best, NP and possibly dissolved
Nanoparticles (NPs) remain an important emerging contaminant in the environment and are much studied.1,2 Their uses, and therefore potential discharges, are large and rapidly growing.3,4 For many NPs such as silver, ceria, zinc oxide, iron oxide, and iron, concentrations are likely to be in the ppt to ppb range in surface waters but are increasing rapidly.5 At these concentration ranges, adverse biological effects may be observed.6 However, in many cases toxicity is only observed at higher concentrations,7,8 whereas lower concentrations may give rise to subtle biological effects without showing obvious signs of toxicity.9 The question of NP toxicity becomes more complicated when considering the dynamic nature of NP transformations in complex media, such as exposure media, which are dependent on both media composition and NP concentration.10−12 This complicates the interpretation of toxicity data and understanding of the dose−response relationship. Due to the lack of appropriate methodologies,13,14 there remains a significant knowledge gap in dosimetry, where the quantification of exposure and dose in NP toxicity testing is © XXXX American Chemical Society
Received: September 25, 2017 Revised: December 15, 2017 Accepted: January 26, 2018
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DOI: 10.1021/acs.est.7b04968 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
exposure concentrations at 1:1 and 1:3 ratios for analytical simplicity, in volumes of 30 mL and exposed to either dissolved Au at concentrations of 1, 2, and 3 ppb or Au NPs (60 nm NPs; NIST 8013) at concentrations of 200 000 and 600 000 particles mL−1. The cell concentrations are somewhat higher than environmental concentrations, but appropriate to validate the technique. The cell cultures and exposures were performed in an incubator (ThermoFisher) to allow for good control over light exposure and temperature. Each exposure study was run in triplicate at 20 °C for up to 77 h with a 12:12 h light:dark cycle, again modified from standard protocols for analytical simplicity. The cells were counted daily using a hemocytometer to check the cell growth for uptake and elimination experiments (Growth curves can be found in Supporting Information (SI) Figures S1 and S2). During the exposure, 1 mL aliquots were removed periodically for analysis, without substantially changing the original volume. Prior to analysis, the cells were separated from the exposure media and washed with media three times to ensure SC-ICP-MS measurements were of cellular and not media concentrations. The cells were centrifuged for 15 min at 300 g and resuspended in 1 mL of fresh culture media, containing no NP or ionic Au (the wash procedure can be found in SI Figure S3). After the three washes, the cells were recounted by hemocytometer and the cell recovery was found to be 43.8 ± 8.6% after the wash cycles. The exact recovery for each sample was back calculated and a correction factor was added to each sample. For the elimination study, the cell culture was initially exposed to 600 000 NPs mL−1 for 22 h. At this point the cell culture was washed by gentle centrifugation (300g for 15 min) and resuspended in fresh media multiple times, each time replacing with fresh media. This resulted in a cell pellet which was resuspended in fresh media a final time without NP addition. The cell culture was then allowed to grow under the same conditions (12:12 light:dark cycle at 20 °C) for a further 55 h in media with no NPs present. One mL aliquots were removed at several time points, treated identically to the exposed cells and analyzed. The supernatant samples from the first and final wash steps were retained and centrifuged at 1000g for 5 min to remove any excess algae. These were then analyzed for dissolved Au and Au NP concentrations. The supernatant from the first wash was analyzed for NP size and number, to assess if any NP transformations have occurred or changes in the exposure concentration during the exposure time period, whereas the final wash was analyzed to check there was no measurable amounts of Au in either dissolved or NP forms in the media which contained the washed cells (SI Figure S4-A and -B for dissolved and NP Au respectively). All results were compared to control samples of media, unexposed cells and the exposure metal in the media (SI Figure S5). The supernatants after each wash step were also collected and measured to ensure complete removal of the exposure metal). ICP-MS Measurements. All analyses were carried out on a PerkinElmer NexION 350 D ICP-MS with a 2 mm quartz injector and quartz torch operating with an RF power of 1600 W. Transport efficiencies were calculated and measured using both 60 nm NIST Au (8012) and lanthanide-doped polystyrene beads with a diameter of 2.5 μm. The latter were used as analogous to cells in cell density and size.
concentrations of metals are measured in exposure media and the total metal concentration of a large number of cells is measured and averaged over the entire cell population to estimate the cellular concentrations.23 For instance, acid digestion of samples followed by inductively coupled plasma mass spectrometry (ICP-MS) measurements give such data. In addition, microscopy measurements have been used to give qualitative information on NP cellular uptake24 on an individual cell basis, but are prone to artifacts from sample preparation, lack of specificity and beam damage, are time-consuming, require extensive sample preparation (electron microscopy) and in some cases lack spatial resolution (most types of light microscopy). Accurate quantification of NP (and metal) concentrations within cells on a single-cell basis has been tried but cell lysis and lack of quantification remains a problem,25,26. However, such data are required to further elucidate bioaccumulation and toxicity behavior of NPs (and metals), or assessment the potential hazards and risks of NPs in the environment.27,28 Some previous attempts of single cell ICP-MS have been made25,26 and report that faster dwell times and more consistent transport efficiency of the cells into the plasma were necessary for full quantitative analysis. These attempts look for correlations between nutrient metals in cells with low correlation coefficients between peak numbers. All previous reports lack validation of the cell viability or transport efficiency. In this work, we present single cell ICP-MS (SC-ICP-MS), a technique capable of delivering intact individual cells into the ICP-MS plasma and quantifying the metal content associated with each cell down to a few attograms (ag; 10−18 g) per cell. This technique allows the nutrient metal concentration as well as metals or NPs taken up or strongly sorbed to the cell to be quantified. The paper has two components. The first is concerned with validating a new introduction system, ensuring that individual intact cells reach the plasma and that the number of cells and the mass of metal per cell can be correctly quantified. For this we compared the transport efficiency (TE) of both nanoparticles (monodisperse 60 nm Au NIST NPs) and micrometer (2.5 μm lanthanide-doped polystyrene beads) sized particles into the ICP-MS using both of the baffled cyclonic and Asperon introduction systems. The micrometersized beads are of a similar size and density to cells, making them an appropriate standard for cells. They were also used to validate that these larger objects were fully atomized and ionized in the plasma by quantifying the mass of lanthanide metals embedded within them. The second part of the paper is concerned with applying the technique to algal cells exposures, including data showing that the algae are not lysed by the sample introduction system. SC-ICP-MS was used to quantify the mass of Au metal strongly associated (either internalized or strongly bound to the cell wall) with fresh water algae (Cyptomonas ovate) for both nanoparticle and dissolved metal exposures.
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MATERIALS AND METHODS Samples and Sample Preparation. The freshwater algae Cyptomonas ovate was used in all exposures using modified OECD protocols.29 Cyptomonas ovate is a flagellated algal species of 20−30 μm in size and chosen after validation of transport through the spray chamber, (see later discussion). Cell cultures were prepared at concentrations of 200 000 cells mL−1, higher than OECD values but chosen to match the NP B
DOI: 10.1021/acs.est.7b04968 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology ICP-MS of Digested Samples. The sample introduction system consisted of a baffled cyclonic spray chamber and concentric glass nebulizer (Minehard) with a nebulizer gas flow rate of 0.92 mL min−1 under standard conditions for Ce140, Eu151, Eu153, Ho165, and Lu175. SP-ICP-MS Measurements of Polymer Beads and NIST Au. The samples were introduced to the plasma using a baffled cyclonic spray chamber and concentric nebulizer (Minehard) were used for sample introduction; a nebulizer gas flow rate of 0.92 mL min−1, and sample flow rate of 0.283 mL min−1 under standard conditions (Table 1).
5 mL or hydrogen peroxide (optima grade) and 10 mL of nitric acid (optima grade). They were left open for 10 min to allow gases to be released from any initial reactions and then resealed. The beads were then digested in a Titan MPS Microwave (PerkinElmer). The conditions table is in SI Table S1. They were allowed to cool overnight after the reaction had taken place and then diluted to 2% acid for analysis. Standards of 10, 50, 100, and 200 ppb Lu, Eu, Ho, and Ce were matrix matched to the samples for ICP-MS analysis. Ge and In were spiked into the samples as internal standards. Cell Viability. The viability of different sized cells was examined to ensure the cells enter the plasma without lysis. For this, three strains of algae were used (Chroomonas sp, Cryptomonas ovata, and Gonyostomum semen) of varying sizes (5−7 μm, 20−30 μm, and 50−70 μm, respectively). The cells were aspirated through the nebulizer at a flow rate of 100 μL min−1 at nebulizer gas flows of 0.2−0.5 mL min−1.
Table 1. Experimental Conditions Used and Transport Efficiencies (TEs) Measured for NIST Au Standards and Lanthanide-Doped Polystrene Beads spray chamber nebulizer spray chamber path neb gas flow (mL min−1) make up gas (mL min−1) sample flow rate (mL min−1) dwell time (μs) sample analysis(s) sample required for analysis (μL) TE 60 nm NIST (%) number of 60 nm NIST NPs measured in a 1 min scan TE 2.5 um beads (%) number of 2.5 um beads measured in a 1 min scan
baffled cyclonic
asperon
glass concentric cyclonic 0.92 0 0.283 50 60 800- 1000 2.42 (±1.85) 342.3 (±8.7)
high efficiency glass concentric liner pass 0.32 0.7 0.015 50 60 100−200 31.33 (±2.54) 229.5 (±19.1)
0.04(±0.02) 3.5 (±1.9)
30.31(±1.85) 208.3(±12)
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RESULTS AND DISCUSSION For the accurate quantification of metal concentrations within cells on a single cell basis to be achieved certain parameters need to be optimized; intact individual cells need to be introduced into the plasma, cells must be fully atomized and ionized and the transport of ions, NPs and micrometer-sized objects must be the same. Standard introduction systems such as the baffled cyclonic (SI Figure S7-A) or parallel path chambers are designed to allow fine droplets30 (
