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In Situ Sensing of Reactive Oxygen Species on Dye-Stained Single DNA Molecules under Illumination.


ABSTRACT: Reactive oxygen species (ROS) are a necessary evil in many biological systems and have been measured with fluorescent probes at the ensemble levels both in vitro and in vivo. Measuring ROS generated from a single molecule is important for mechanistic studies, yet measuring ROS near a dye-labeled single-molecule under illumination has been challenging. Here, we use CellROX, a group of ROS probes, to sense ROS near dye-stained DNA that has been flow-stretched and immobilized on a surface. ROS is responsible for the photodamage of DNA molecules under this circumstance. In this report, we confirmed the ROS sensing reaction in bulk solutions and optimized the conditions for single-molecule experiments including the selection of substrates, dye concentrations, probes in the CellROX series, excitation lasers, and emission filter-sets. We observed a correlation between ROS and the dye-labeled DNA and localized the ROS-activated CellROX probe molecules at both the ensemble level and the single-molecule level.

SUBMITTER: Pyle JR 

PROVIDER: S-EPMC6813813 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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In Situ Sensing of Reactive Oxygen Species on Dye-Stained Single DNA Molecules under Illumination.

Pyle Joseph R JR   Sy Piecco Kurt Waldo E KWE   Vicente Juvinch R JR   Chen Jixin J  

Langmuir : the ACS journal of surfaces and colloids 20190820 35


Reactive oxygen species (ROS) are a necessary evil in many biological systems and have been measured with fluorescent probes at the ensemble levels both in vitro and in vivo. Measuring ROS generated from a single molecule is important for mechanistic studies, yet measuring ROS near a dye-labeled single-molecule under illumination has been challenging. Here, we use CellROX, a group of ROS probes, to sense ROS near dye-stained DNA that has been flow-stretched and immobilized on a surface. ROS is r  ...[more]

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