*5.1. Detection of GFNs in Cells and Organism Tissues*

The detection of GFNs internalization (distribution and behavior) in model organisms and cells is a key step for a better understanding of their genotoxicity and underlying mechanisms. The most commonly used detection technique includes direct observation of localization of GFNs in organisms and cells by transmission electron microscopy (TEM) [88]. The hyperspectral imaging is also used to visualize cellular interactions with NPs [134], such as cellular uptake and binding of GFNs [87]. The label-based approaches to image GFNs exist in cells by confocal and fluorescence microscopy, reflection-based imaging, and flow cytometry. Additionally, scanning electron microscopy (SEM) can be used to detect the attachment of GFNs in the surface zone of cells [52,87]. Raman spectroscopy and atomic force microscopy (AFM) were used to evaluate nuclear area changes and the disruption of DNA chains impacted by GQDs, respectively [69]. However, these traditional techniques are limited by low observation efficiency and large errors of quantitative results, with are disadvantages in the detection of GFNs [88]. Few studies focus on GFNs nuclear detecting techniques. In the biological imaging field, most research pays attention to safe application of fluorescent GFNs nuclear images rather than assessing genotoxicity of GFNs from an environmental toxicology point of view [135–137]. It is necessary to further optimize and develop detection techniques of GFNs in cells and organism tissues for a better understanding of genotoxicity. For example, Chen et al. [138] used laser desorption/ionization mass spectrometry imaging to map and quantify precisely the sub-organ distribution of the carbon nanotubes, GO, and carbon nanodots in mice. The SEM–Raman spectroscopy co-located system provide both SEM and Raman data from the

same area on the cell sample, which avoids sample registration issues and makes observed results more accurate [139].

#### *5.2. Genotoxicity Assay of GFNs*

There are several assays available to access the genotoxicity of GFNs, measuring various endpoints [98]. The Ames test (bacterial reverse mutation), the comet assay (single cell gel electrophoresis), the chromosomal aberration (CHA), and micronuclei (MN) are the most common tests for genotoxicity. The Ames test (bacterial reverse mutation) can provide initial testing for genotoxicity. The comet assay can detect DNA damage, while the CHA and MN can test large chromosomal abnormalities. The hypoxanthine phosphoribosyl transferase (HPRT) gene is suitable for assessing mutations induced by suspect genotoxic agents, such as NPs [98]. Oxidative DNA damage should be considered one of the causes of genotoxicity. Superoxide radicals can lead to the activation of oxidation of the guanine bases present in the DNA strands, causing rupture to these strands. The most commonly used detection techniques include 8-hydroxydeoxyguanosine and 7, 8-dihydro-oxodeoxyguanine by HPLC with electrochemical detection [140].
