2.4 Quantification of intracellular ROS
Bacterial intracellular ROS level was measured using
2’,7’-dichlorofluorescent diacetate (DCFH-DA) as
the fluorescent
probe.[22] Briefly, the E. coli or S.
aureus in logarithmic growth phase was centrifuged and washed with PBS.
Next, 100 μL of the bacteria suspended in PBS (OD600 nm= 0.2) was mixed with the CD solution (100 μL) at a predetermined
concentration in a 96-well plate. Subsequently, the 96-well plate was
exposed to UV irradiation for 30 min. The suspension was then spiked
with DCFH-DA at a final concentration of 10 μM, followed by incubation
for another 30 min in the dark. The fluorescence intensity at 525 nm of
the mixture was measured at an excitation wavelength of 496 nm using the
microplate reader. The change in ROS level was assessed using the rate
of increase (F/F0 ) in the fluorescence intensity
upon incubation with the CDs, in which F is the fluorescence
intensity of the system containing the CDs and F0is that without incubating with the CDs. A bacterial suspension to which
1 mM of H2O2 was added instead of the
CDs was used as a positive control. In parallel, the bacterial
intracellular ROS levels upon addition of the CDs in the dark were
evaluated.