Almost every industry has discovered ways in which nanomaterials can improve the functionality of their products. While useful, nanomaterials are often considered to be uncharted territories in terms of their potential toxicity to both humans and the environment.
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Recent work has found that impedance flow cytometry has the potential to accurately measure specified properties of nanomaterials in cell suspensions to determine their potential toxic effects.
Emerging nanotoxicity concerns
Nanomaterials are associated with several advantageous physicochemical properties that have led to their widespread use in a number of industrial applications, some of which include medicine, biotechnology, electronics, food, agriculture and energy, to name a few.
The individuals manufacturing and handling nanomaterials are therefore frequently exposed to these materials frequently, which can lead to adverse health effects on both these humans and the environment to which the materials are being released. As a result, the field of nanotoxicology has emerged to address the potentially toxic effects associated with acute and chronic exposure to nanomaterials.
Limitations of current nanotoxicology testing strategies
Despite the apparent need to perform in depth toxicological studies on nanomaterials, there remains a lack of standardized techniques capable of evaluating the physicochemical properties of these materials and their potential health effects.
For example, pharmaceutical and chemical industries have often relied upon colorimetric in vitro assays for nanotoxicology reports; however, nanomaterials have been shown to interfere with various stages of these experiments and ultimately generate either false-positive or false-negative results.
These nanomaterial-caused interferences have been found to be specific to the particle, concentration and assay, thereby resulting in the need for highly specific and optimized in vitro assays to be developed for each type of nanomaterial, which is an incredibly tedious and costly task for these industries to perform.
Flow cytometry and nanotoxicology
Although flow cytometry is capable of providing accurate cell counts in a rapid manner, the need to label cells prior to conventional flow cytometry analysis can lead to unwanted interactions between these labels and nanomaterials.
Impedance flow cytometry, on the other hand, is a label-free and impedance-based flow cytometry method that can provide similar multiparametric information on cells, thereby demonstrating its applicability for nanotoxicology studies.
At the end of an impedance flow cytometry run, data is provided in the form of the electric impedance (Z) of the cell analytes, which represents both the resistance (R) and the reactance (X), the latter of which is dependent on frequency.
Understanding impedance flow cytometry analysis
In order to obtain these values, the impedance flow cytometry instrument relies on an electric field-based analysis that monitors the behavior of the analyte when placed in an electric field at varying frequencies. At low frequencies, for example, the barrier of the cell membrane resists the current flow, which enables the user to obtain information on the cell size.
As the frequencies rise to an intermediate level, more information on the specific properties of the cell membrane can be obtained. Subsequently, high frequencies can penetrate the cell membrane to provide information about the interior components of the cell.
To date, impedance flow cytometry instruments have been enhanced by the technology of Switzerland-based company Amphasys AG, which has developed a portable IFC instrument that is now available for commercial use.
Within the Amphasys AG system, a microfluidic chip provides data through the use of two microelectrodes present on each side of the microchannel. Whereas one pair of the electrodes are used to provide reference data, the other microelectrode pair senses any change that arises in the electric current as a result of the cell passing through the microchannel.
All cell measurements, which reflect the change in impedance that results from the cell’s movement, are provided in the form of a density scatter plot.
Testing impedance flow cytometry for in vitro nanotoxicity studies
A recent study published in Scientific Reports explored the reliability and applicability of impedance flow cytometry for in vitro nanotoxicity screening.
In their work, the relevant physicochemical properties of eight different nanomaterials were assessed by initially creating an nanomaterial dispersion that was subsequently placed in U937 human histiocytic lymphoma cells at varying concentrations of 2, 10, 20, 50 and 100 microgram (µg)/milliliter (mL).
The impedance flow cytometry instrument used to analyze the cell samples in this study was the Zetasizer Nano ZSP, which is manufactured by Malvern Instruments Ltd. All impedance flow cytometry data were compared to those obtained by both a trypan blue (TB) dye exclusion assay and conventional flow cytometry .
The current study found that while both the TB assay and impedance flow cytometry eliminated the occurrence of any nanomaterial-cased interferences, impedance flow cytometry could provide more information on the intracellular density and cell size of samples that was not possible with the TB assay.
As expected, the interactions between the nanomaterials and the dyes used in the conventional flow cytometry method led nanomaterial-specific artifacts to arise, thereby discrediting this method for accurate cell analysis.
Conclusion
In addition to providing more information than the TB method, impedance flow cytometry analysis can also measure up to 10,000 cells, which is comparable to the 200-300 cell capability of the TB assay.
The ability to analyze a greater number of cells not only improves the reliability of impedance flow cytometry, but also allows smaller subpopulations of cells to be analyzed that would otherwise not be possible when the TB assay is employed.
Furthermore, the Zetasizer Nano ZSP system utilized in this study is a highly versatile tool that can be further enhanced with microfluidic chips of varying sizes for a more robust and sensitive analysis.
In addition to demonstrating its usefulness in studying nanomaterial-induced toxicity, future application of impedance flow cytometry is expected to assist in the toxicological analyses of larger particles, chemicals and pharmaceutical agents.
Sources
Ostermann, M., Sauter, A., Xue, Y., Birkeland, E., Schoelermann, J., et al. (2020). Label-free impedance flow cytometry for nanotoxicity screening. Scientific Reports 10(142). doi:10.1038/s41598-019-56705-3.
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