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Multifaceted fluorescent system gives finer measure of hypoxia

Probes tuned to different oxygen levels allow researchers to measure hypoxia with higher accuracy in living cells

by Alla Katsnelson, special to C&EN
January 31, 2023

A grid of images showing how two hypoxia change the color of cancer cells as oxygen levels fall. One probe changes color at 4% oxygen, the other changes color at about 0.5% oxygen.
Credit: J. Am. Chem. Soc.
By using two hypoxia probes in tandem, researchers can observe the oxygen levels around these lung cancer cells. Probe 1 gives a fluorescence signal when oxygen concentration reaches 4%. Probe 2 fluoresces a different color when cells encounter an even lower oxygen concentration. The bars in the lower right corner of each square represent 20 µm.

Cells live and die by the presence of oxygen, and low-oxygen states often indicate disease. Bacterial biofilms and the cells in solid tumors, for example, are hypoxic—that is, have abnormally low oxygen levels. But current methods to detect hypoxia are cumbersome and provide limited information, often only giving a yes or no answer.

Stuart Conway, an organic chemist and chemical biologist at the University of Oxford, and his colleagues had previously developed tools to deliver cancer drugs selectively to hypoxic cells, but they needed a way to recognize where to direct them in the dynamic and ever-changing tumor microenvironment. By harnessing the redox sensitivity of indolequinones, they created a set of fluorescent probes that can be combined to image different degrees of hypoxia (J. Am. Chem. Soc. 2023, DOI: 10.1021/jacs.2c12493).

In living cells, indolequinone is readily reduced in an enzymatic reaction, losing an electron to produce a radical anion. And that radical can bounce back to its starting structure if it encounters oxygen. Conway and his colleagues figured they could visualize this back-and-forth reaction by attaching a fluorescent molecule to the indolequinone that shines only when indolequinone gets reduced and releases the fluorophore. This created a push-pull relationship between oxygen levels and the fluorescence the researchers could detect.

A reaction scheme showing a fluorescent molecule breaking off a larger molecule when oxygen concentration drops below 4%.
As oxygen concentration drops, this probe breaks apart and releases a molecule with orange fluorescence.

The researchers narrowed the range of sensing using the chemical properties of multiple fluorescent molecules. A molecule with a strong leaving group will pop off even at relatively high oxygen concentrations, while a weaker leaving group will be released only if the amount of oxygen present is a relatively low. Conway says that means it’s possible to link the ability of the leaving group to leave with the level of oxygen that’s in the cells’ environment. Upon release, the fluorophores would be activated, and the colors detected by live cell imaging would reflect the oxygen concentrations of the cells.

The group tested multiple fluorophores in both 2D and 3D cell culture systems and homed in on two that would not kill the cells and fluoresced predictably when triggered by specific oxygen levels. One began glowing red when oxygen levels dropped to 4%, and the other only showed its green hue at oxygen levels under 0.5%.

It may be possible to use the fluorescence probes in a living organism, too, says Conway, but light must penetrate the tissue for them to work. The team is now adapting the approach to work with magnetic resonance probes instead of fluorescent ones, which would make it more amenable to using it inside a living organism. By deploying the probes in the body, researchers could find and target hypoxic tumor cells for treatment.

“This work provides an innovative activity-based sensing approach” to sensing different hypoxia levels, says Christopher J. Chang, a chemical biologist at the University of California, Berkeley, who was not involved in the work, in an email. Combining a hypoxia-sensitive trigger with carefully tuned fluorophores “offers a broadly useful approach for gradient detection of other biological analytes,” he says.


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