Photodamage and the Importance of Photoprotection
in Biomolecular-Powered Device Applications
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Abstract
In
recent years, an enhanced understanding of the mechanisms underlying
photobleaching and photoblinking of fluorescent dyes has led to improved
photoprotection strategies, such as reducing and oxidizing systems
(ROXS) that reduce blinking and oxygen scavenging systems to reduce
bleaching. Excitation of fluorescent dyes can also result in damage
to catalytic proteins (e.g., biomolecular motors), affecting the performance
of integrated devices. Here, we characterized the motility of microtubules
driven by kinesin motor proteins using various photoprotection strategies,
including a microfluidic deoxygenation device. Impaired motility of
microtubules was observed at high excitation intensities in the absence
of photoprotection as well as in the presence of an enzymatic oxygen
scavenging system. In contrast, using a polydimethylsiloxane (PDMS)
microfluidic deoxygenation device and ROXS, not only were the fluorophores
slower to bleach but also moving the velocity and fraction of microtubules
over time remained unaffected even at high excitation intensities.
Further, we demonstrate the importance of photoprotection by examining
the effect of photodamage on the behavior of a switchable mutant of
kinesin. Overall, these results demonstrate that improved photoprotection
strategies may have a profound impact on functional fluorescently
labeled biomolecules in integrated devices