Observing the Reversible Single Molecule Electrochemistry of Alexa Fluor 647 Dyes by Total Internal Reflection Fluorescence Microscopy

  • Angew Chem Int Ed Engl. 2019 Oct 7;58(41):14495-14498. doi: 10.1002/anie.201907298.
Sanjun Fan  1 ,  James E A Webb  1 ,  Ying Yang  1 ,  Daniel J Nieves  2 ,  Vinicius R Gonçales  1 ,  Jason Tran  2 ,  Geva Hilzenrat  2 ,  Mohaddeseh Kahram  1 ,  Richard D Tilley  1 ,  Katharina Gaus  2 ,  J Justin Gooding  1
Affiliations
  • 1. School of Chemistry, Australian Centre for NanoMedicine and The ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of New South Wales, Sydney, NSW, 2052, Australia.
  • 2. EMBL Australia Node in Single Molecule Science and ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney, NSW, 2052, Australia.
PMID: 31418504 DOI: 10.1002/anie.201907298
Abstract

Alexa Fluor 647 is a widely used fluorescent probe for cell bioimaging and super-resolution microscopy. Herein, the reversible fluorescence switching of Alexa Fluor 647 conjugated to bovine serum albumin (BSA) and adsorbed onto indium tin oxide (ITO) electrodes under electrochemical potential control at the level of single protein molecules is reported. The modulation of the fluorescence as a function of potential was observed using total internal reflectance fluorescence (TIRF) microscopy. The fluorescence intensity of the Alexa Fluor 647 decreased, or reached background levels, at reducing potentials but returned to normal levels at oxidizing potentials. These electrochemically induced changes in fluorescence were sensitive to pH despite that BSA-Alexa Fluor 647 fluorescence without applied potential is insensitive to pH between values of 4-10. The observed pH dependence indicated the involvement of electron and proton transfer in the fluorescence switching mechanism.

Keywords
Alexa Fluor 647; electrochemical switchable fluorescence; electron transfer; proton transfer; single molecules.
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