Hello. I am working on ROS production of two systems:
system A is cerium oxide and hydrogen peroxide, system B is cerium oxide nanoparticle, hydrogen peroxide and potassium bromide. I did some antibacterial tests before and the bacteria(e.coli) cultivate in LB broth later mixed with these two systems were killed up to 100%. The results were obtained by plate couting and the plate with system B can kill all E.coli every time, while system A can kill most of them, so I want to go further and investigate the ROS production: singlet oxygen and hydroxyl radical.
singlet oxygen: excitation 488nm, emission 528nm;
hydroxyl radical: excitation 476nm, emission 516nm.
I used a clear-bottom black 96 well plate and add 100ul probe in each well to mix with the systems as below, then incubate them under room temperature for 30 min in dark before using the microplate reader:
system A(CeO2+H2O2)+E.coli
system B(CeO2+H2O2+KBr)+E.coli
system A(CeO2+H2O2)
system B(CeO2+H2O2+KBr)
The result was strange, fluorescence of system A+E.coli sometimes even bigger than system B+E.coli, and the result of system A or B without E.coli addtion were negative values, or bigger than with E.coli addition.
I think there must be some contamination in the process, and I am not familar with all the setting of the microplate reader.
Please help me........
Based on your excitation/emission wavelength, I guess that you have add fluorescent probes for 1O2 ans HO°.
Second 1O2 ans HO° are transient species. Only a small fraction will reach the E.coli. This can justify the same signal with/without E.coli.
The question is do you detect more ROS in system B or A ?
Robert Bernard Pansu Thank you so much for your reply!!! Unfortunately in our lab we only bought these two kits for ROS detection, so I haven't get a chance to test other ROS species...
Robert Bernard Pansu Sorry for the picture is not clear. The data is the fluorescence signal: 1-4 are system A/B+E.coli, 5-8 are A/B control.
The frames means the excitation and emmision for singlet oxygen/hydroxyl radical. The raw data shows that system B could produce more than system A but I don't think these results are consistent, one of phd students in our lab told me that if the one of the result could meet my expectation, which means it could be true so I just need to repeat few more times...I think I should add more control groups but I am not sure.
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