Environmental Engineering Reference
In-Depth Information
for the detection of ENPs in environmental samples is one of the major hurdles in mitigat-
ing their adverse health impact. The irst uncertainty is in the representation of the con-
centration of the ENPs. Different metrics such as mass (μg/mL), number (10
n
particles/mL),
or surface area (μg /c m
3
) have been used to express the concentration of the ENPs, which
makes it dificult to correlate the existing data [36].
At present, the commonly used methods for the detection of uptake of ENPs in the cells
are transmission electron microscopy (TEM), scanning electron microscopy along with
backscattered electron and energy-dispersive x-ray spectroscopy (SEM + BSE + EDS), con-
focal and luorescence microscopy, relection-based imaging, and low cytometry [37,38].
These techniques enable the tracking of ENPs in the cells as well as cellular organelles. The
high resolution of TEM enables the imaging of membrane invagination, mode of ENPs'
uptake, and ultrastructural changes occurring in the cells subsequent to ENP treatment.
SEM, on the other hand, is used to study the morphological changes and ENP interactions
with the cell. On the other hand, EDS coupled with SEM provides an additional feature
to analyze the elemental composition of the specimen based on the released energy by
the corresponding element [38,39]. Although these imaging techniques provide several
advantages, there are certain drawbacks; for example, in TEM and SEM, the samples have
to be ixed; therefore, live cell uptake cannot be monitored. It is also resource intensive,
time consuming, and conined to imaging of a few cells. Furthermore, the staining pro-
cess introduces electron-dense artifacts that may be construed as nanoparticles. Confocal
and luorescence microscopy, on the other hand, require that the particles be tagged with
a probe or be doped with a luorescence dye for their detection. Since the native nature of
ENPs is lost, it can lead to false/incorrect interpretation of observations [39].
Flow cytometry is another technique used to assess the uptake of ENPs in the cells. This
is a rapid, multiparametric, single-cell analysis with robust statistics, owing to the large
number of events measured per treatment, and is cost-effective [40]. In this method, a laser
beam strikes on the stream of luid containing single-cell suspension. The light diffracted,
relected, and refracted by the cells is recorded by the photomultiplier tubes and the elec-
tronics convert these optical pulses to digital values. It is well established that the light
diffracted by the cells represents the forward light scatter and is used to measure the cel-
lular size. However, the relected and refracted light corresponds to the side scatter, which
is a combined effect of the granularity and the cellular mass of the cell. ENPs in the host
cell serve as granules and relect/refract the light based on their intrinsic property. As the
ENPs enter into the cells, the side-scatter intensity of the cell increases proportionately to
the concentration of the ENPs [37,41]. A luorescent particle can give an increased signal
of side scatter as well as the luorochrome intensity in a dose-dependent manner. Most
aerobic microbes divide quickly under optimal growth conditions (viz.
E. coli
in 20 min);
thus, the internalization of the ENPs in the cells and their retention for several genera-
tions can easily be monitored in a short time using low cytometry. Several studies have
demonstrated the internalization of the ENPs in cell lines using low cytometry [37,41-43].
Using the aforementioned techniques, it has been shown that ENPs can be internalized in
a number of aquatic organisms (Figure 5.2).
Despite having several techniques to track the ENP uptake in different aquatic organ-
isms, the precise mechanism of uptake is still unknown. However, it has been proposed
that nonspeciic diffusion, nonspeciic membrane damage and speciic uptake (through
porins), phagocytosis, and membrane ion transporters are the possible mechanisms
through which the ENPs could pass through the cell wall and membranes of different
aquatic organisms [8,44].
