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Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation
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Fluorescence-enabled electrochemical microscopy with dihydroresorufin as a fluorogenic indicator.

Stephen M Oja1, Joshua P Guerrette, Michelle R David

  • 1Department of Chemistry, University of Washington , Seattle, Washington 98195-1700, United States.

Analytical Chemistry
|May 30, 2014
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Summary

We expanded fluorescence-enabled electrochemical microscopy (FEEM) to study cathodic reactions using dihydroresorufin. This new indicator allows for improved temporal response and reduced signal broadening in electrochemical imaging.

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Area of Science:

  • Electrochemistry
  • Analytical Chemistry
  • Microscopy

Background:

  • Fluorescence-enabled electrochemical microscopy (FEEM) is a novel technique for electrochemical imaging.
  • FEEM couples electrochemical reactions to fluorogenic reactions for signal conversion.
  • Previous FEEM applications focused on oxidation reactions using resazurin.

Purpose of the Study:

  • To extend FEEM capabilities to the study of cathodic (reduction) reactions.
  • To introduce and evaluate dihydroresorufin as a new fluorogenic indicator for FEEM.
  • To demonstrate quantitative detection of reducible analytes using FEEM.

Main Methods:

  • Utilized a closed bipolar electrode system to couple electrochemical and fluorogenic reactions.
  • Employed dihydroresorufin, a nonfluorescent precursor oxidized to fluorescent resorufin.
  • Performed potential sweep experiments and simultaneously recorded fluorescence and current.
  • Applied FEEM to detect ferricyanide reduction at an ultramicroelectrode.

Main Results:

  • Successfully adapted FEEM for cathodic reactions using dihydroresorufin.
  • Demonstrated quantitative detection of ferricyanide down to approximately 100 μM.
  • Observed improved temporal response and reduced diffusional broadening with dihydroresorufin compared to resazurin.
  • Correlated fluorescence intensity with electrochemical oxidation current.

Conclusions:

  • Dihydroresorufin is an effective indicator for studying cathodic reactions with FEEM.
  • FEEM, enhanced by dihydroresorufin, offers improved performance for electrochemical imaging.
  • The technique enables sensitive and quantitative detection of reducible analytes.