Hello everyone,
I'm studying NPs-induced oxidative stress in the marine microlaga P. tricornutm, using the fluorescent probe DCFH-DA to measure ROS production. Briefly, I am incubating my cells with 10 uM dichlorofluorescin for 60 min in dark before removing the medium and exposing the cells to different concentrations of carboxylated polystyrene NPs. Then I measure the probe fluorescence every 5 min over 1 hour, according to Wang & Joseph (1999) and Aranda et al. (2013) - both attached - calculating the fluorescence increase over time of exposed vs. control groups.
I've tried out this method several times and it works smoothly (hydrogen peroxide postive controls are nice). I have not observed a ROS increase in cells exposed to this kind of NPs, as expected. However, I've noticed that fluorescence intensity slightly decreased, in a conc.-dependent fashion, with increasing NP concentrations. I'm pretty sure that this small fluorescence decrease would not mask anyway an ongoing oxidative stress in exposed cells, yet I am curious if anybody ever experienced DCFH-DA fluo qunenching with this kind of nanoparticles. Has anybody had the same issue? Any ideas on how to circumvent it? I know NP interference with fluorimetric/colorimetric test is an issue sometimes but I could not find anything about this NP type.
Thanks a lot for your suggestions
Yes! there is a likelihood of the fluorescence quenching (direct or indirect) by nanoparticles. Some of the nanoparticle like one I used, had the intrinsic radical scavenging ability and thus reduced the amount of ROS in the system and thus decreased the fluorescence (perhaps this was not quenching but results were apparent as quenching), So this may also be the case with your particles, secondly the direct interaction of some of the inorganic electron active regions of nanoparticles can cause quenching. Changing the solvent system (if possible for your experiment) may help. Moreover, the modification or blocking of specific groups may lead to the suppression of quenching. Moreover, if they really quench, it could be a useful tool for designing probes for some techniques.
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