Detecting Trace Elements in Single Cells with ICP-MS

Simon Nelms- ICP-MS Product Marketing Manager, Thermo Fisher Scientific

Trace elements are crucial to many biological processes. From the wound-healing effects of zinc to the enzymatic control of iron and copper, and the potential anti-cancer benefits of selenium, trace elements are critical to physiological and biochemical processes in plants and animals.

To fully understand trace element uptake and processing, intracellular levels of trace elements must be determined. By detecting both the distribution and mass concentration of trace elements within cell populations, scientists can gain greater insight into cellular function and heterogeneity within populations. This knowledge can help:

• Improve understanding in clinical research, through the detection of metal toxins.
• Drive better drug efficacy, by detecting metallodrug uptake.
• Define optimal cell culture conditions for bio-production research, by analyzing consistency markers within cultures.

Although biomarker analysis is routinely used in clinical research, diagnosis and treatment, trace metal analysis has lagged. This is largely because traditional methods, which rely on cell digestion, assume homogeneous distribution of analytes through the cell cohort. This is not the case with trace elements and these methods do not give the detail needed to discern the nuances of intracellular distribution.

However, recent advances in inductively coupled plasma-mass spectrometry (ICP-MS) are changing this landscape. These powerful, element-selective detection systems now provide the capacity and capability for trace element analysis at the single-cell level. This paper shows how scientists can use the latest ICP-MS technology and software to accurately determine the amount of trace selenium in individual cells using certified reference samples of selenized yeast.

What is Single-Cell Analysis and Why is it Important?

As the title suggests, single-cell analysis is the determination of trace elements in individual cells. Understanding trace element presence and concentration at the cellular level is imperative as there is often great heterogeneity, even within the same cellular population and in ideal conditions. Without understanding the spectrum of a trace element’s presence and its mass within individual cells, the nuances of its impact on a cell, a population or even a bioculture cannot be fully elucidated.

Traditional trace-element analysis methods involve digesting large numbers of cells and analyzing the metal content within. Although effective at measuring total trace element mass, these methods do not account for cell-to-cell variation and cannot identify the number of cells containing the element or the concentration contained within. This means that valuable insights contained within the cells are lost.

Effective single-cell analysis is needed to accurately quantify the average mass per cell and identify the element distribution across cell cohorts (number concentration). These measurements are established through two main calculations (Figure 1).

Due to recent advances in ICP-MS technology, two key factors in these calculations can now be observed: signal intensity and the number of cell-derived events (shown in red), but the technology and its software also bring other important accuracy benefits. By using optimized workflows and tailored equipment that helps to protect the cells, transport efficiency and detection sensitivity can be improved. Transport efficiencies (for transferring cells from the sample to the plasma source of the ICP-MS) of greater than 70% can now be routinely achieved and high detection sensitivity, (measured through a standardization curve and accounting for element ionization, focusing and detection within the ICP-MS instrument), is achievable for a wide range of elements.

The latest software can also help increase efficiency, improve throughput and lower laboratory costs by automating the user-determined inputs (shown in green). Algorithms built into the software can accurately measure transport and detection efficiencies and use analytical parameters to suggest optimized volumetric flow rates and dwell times to improve these rates.

scICP-MS: Reaching New Levels of Sensitivity and Speed for Single-Cell Analysis

So-called single-cell ICP-MS (scICP-MS) is a technique that utilizes the single-cell mode of the latest ICP-MS technology and can analyze multiple trace elements (in sequential order) from a single sample.

The sample is nebulized to form a stream of single cells for individual analysis. The resulting cells are then ionized, and ion optics focus the beam into a quadrupole mass analyzer where ions, separated according to their mass-charge ratio (m/z), are detected.

The latest scICP-MS technology provides very low detection limits in low sample volumes, increasing sensitivity and allowing even low levels of trace elements to be accurately identified. Added to this, simple sample preparation methods and high throughput workflows mean that a greater number of samples can be analyzed, even at the single-cell level. Part of this efficiency comes from the technology’s ability to control interference and reduce background noise, meaning that analysis of complex cultures requires minimal preparation steps.

However, to achieve high levels of efficiency and throughput while maintaining accuracy, scICP-MS technology must be used in combination with complementary technology and software.

Firstly, specialized nebulizers must be used to decrease the flow rate. This protects the cells from damage and ensures a stream of single cells for individual analysis with high transport efficiency.

Secondly, specialized software helps to drive key elements of the workflow to maximize detection accuracy and efficiency. Decreased dwell times enable detection of the very short, transient signals that are emitted from single cells and sequential analysis programs help to reduce settling times, maximize duty cycles to fully measure m/z, and split sample time equally among analytes (if multi-analyte measurement is needed).

Finally, data packages can automate evaluation parameters for more accurate calculations. Known cell suspension standards can be used to calculate transport efficiency and detection sensitivity can be calculated by calibrating against ionic standards. Once the data is collected, raw data and signal-distribution views can be displayed to accurately identify fractions, modify thresholds and interrogate mass-distribution information. In turn, mean and median analyte mass and number concentration figures can be calculated from this data representation.

Measuring Selenium in Selenized Yeast with scICP-MS

Selenium (Se) is a trace element micronutrient and antioxidant that is thought to protect individuals from thyroid disease, cancer, cardiovascular disease and even cognitive decline.¹ Recent research delivering Se via selenized yeast has shown reduced low-density lipoprotein profiles in patients with atherosclerosis² and reduced recurrence of tumors in patients who initially had an advanced adenoma.³

The bioavailability and low cost of selenized yeast make this a promising supplement and potential treatment for some diseases. However, Se can be present in many different forms in yeast – organic as selenomethionine,⁴ inorganic as selenite or selenate,⁵ and also in nanoparticle form.⁶ To understand the full potential of how selenized yeast might be used in disease prevention and treatment, plus how reliable bioproduction can be maintained, full analyses of the mass and distribution of Se in yeast are crucial.

In a recent experiment, the Thermo Scientific™ iCAP™ TQ ICP-MS triple quadrupole system in single-cell acquisition mode was used to evaluate the presence of Se in a certified reference material sample of lyophilized yeast cells (SELM-1), a type of selenized yeast. Phosphorus (P) was also evaluated as a cell marker, since it is a constituent element in yeast cells, e.g., as a part of the DNA backbone. In this study, the most common isotopes of both elements were measured in their ionic forms: ³¹P+ and ⁸⁰Se+.⁷

By comparing P and Se events as detected through the ICP-MS instrument, the fraction of Se-containing cells could be calculated, as well as the mean and median mass of the analytes within the cell population.

Method

SELM-1 cells were resuspended in water, washed twice by centrifugation and then diluted to a final concentration of 50,000 cells per mL, as confirmed by flow cytometry. Specialized nebulizers and spray chambers were then used to deliver a single stream of cells to the scICP-MS while protecting their delicate structure. High transport efficiencies of greater than 70% were achieved and confirmed by the linked software.

The iCAP TQ ICP-MS was used in TQ-O2 mode to induce oxidation and reduce polyatomic interferences. P and S were measured via the product ions ³¹P+ ¹⁶O+ and ⁸⁰Se+ ¹⁶O+. The exact parameters established for the separation and detection stages are shown in Table 1.

The scQuant plug-in for Thermo Scientific c™ Qtegra™ Intelligent Scientific c Data Solution™ Software was used to create the method and provide data evaluation post-analysis. Data were acquired using the time-resolved analysis mode at a dwell time of 5 ms and a detection sensitivity of 0.2 µgL^-1 was achieved for Se, equivalent to a minimum detectable amount of 0.17 Se fg per cell.

Results

Quantitative assessment of the resulting ICP-MS signals allows the determination of three key metrics: the number of cells that contain quantifiable amount of each element, the average mass of each element within a cell and the distribution of each element across the cell cohort.

The raw data indicates that the number of detected signals per unit of time was slightly lower for Se than for P. This demonstrates that, although each cell contains a significant amount of P (known to be present in DNA), not all cells contain Se and, therefore, only a fraction of the total cells showed detectable levels of Se (Figure 2). In fact, 57% of cells had detectable levels of Se, in the range of 2.5 fg – 72.5 fg.

Further analysis showed a broad distribution of Se in the cell population, a mean of 18.6 fg and a median of 16.8 fg were detected with a standard deviation (SD) of ± 12.5 fg. The mean for P was 37.0 fg and the median 30.9 fg, across the cell population, with an SD ± 23.1 fg (Figure 3). Although the SD was lower for the Se data, it was based only on the detected cells and the overall score would have shown greater inhomogeneity.

scICP-MS is an Effective Tool for Analyzing Trace Elements in Single Cells

This study into the presence of Se and P in selenized yeast demonstrates that scICP-MS, used in single-cell mode, is an effective tool for the analysis of trace elements in single cells. Scientists can now accurately establish trace element mass per cell and the element distribution across a cell cohort.

However, this technique relies heavily on the right analytical software being in place so that detection and transport efficiencies can both be determined accurately, and a sequential approach can be used to measure multiple elements for identical periods on a single sample aspiration. With comprehensive data evaluation tools, both raw and intermediate data can then be utilized to enable full representation and analysis of the mass and number concentration data.

By using scICP-MS technology to detect trace elements at the single-cell level, analysts can gain a greater understanding of the migration and function of trace elements at a cellular level. Data such as these can be used in a variety of applications, perhaps for the development of more advanced therapies for a wide range of clinical presentations, better diagnostics for toxin ingestion or environmental pollution and even to improve quality and efficiency measures in bioproduction processes.

References

1. National Institutes of Health Office of Dietary Supplements. Selenium Fact Sheet for Healthcare Professionals page. Available at: https://ods.od.nih.gov/factsheets/Selenium-HealthProfessional/ Accessed October 19, 2022.
2. Ghazi MKK, Ghaffari S, et al. Effects of sodium selenite and selenium-enriched yeast on cardiometabolic indices of patients with atherosclerosis: A double-blind randomized clinical trial study. J Cardiovasc Thorac Res. 2021;13(4):314-319.
3. Thompson PA, Ashbeck EL, et al. Selenium Supplementation for Prevention of Colorectal Adenomas and Risk of Associated Type 2 Diabetes. J Natl Cancer Inst. 2016;108(12).
4. Klein, EA. Selenium and vitamin E cancer prevention trial. Ann. N.Y. Acad. Sci. 2004;1031:234–241.
5. Gilbert-López B, Dernovics M, et al. Detection of over 100 selenium metabolites in selenized yeast by liquid chromatography electrospray time-of-flight mass spectrometry. J. Chromatogr. B. 2017;1060:84–90.
6. Álvarez-Fernández García R, Corte-Rodríguez, M, et al. Addressing the presence of biologic selenium nanoparticles in yeast cells: analytical strategies based on ICP-TQ-MS. Analyst. 2020;145:1457–1465.
7. Thermo Fisher Scientic. Application Note 001349. Assessing the level and distribution of selenium in selenized yeast cells using single cell ICP-MS analysis. Available at: https:// assets.thermofisher.com/TFS-Assets/CMD/Application-Notes/an-001349-icp-ms[1]selenium-yeast-cells-an001349-na-en.pdf

About the Author

Simon Nelms is a former ICP-OES and ICP-MS applications specialist who’s been part of the Thermo Fisher Scientific team for more than 20 years. He is now the marketing manager for the ICP-MS product range. Simon holds a BSc in Analytical Chemistry and a PhD in research involving ICP-MS method development.

Subscribe to our e-Newsletters
Stay up to date with the latest news, articles, and events. Plus, get special
offers from American Pharmaceutical Review delivered to your inbox!
Sign up now!