Real-time pH microscopy down to the molecular level by combined scanning electrochemical microscopy/single-molecule fluorescence spectroscopy

Frank Mario Boldt, Jürgen Heinze, Manuel Diez, Jan Petersen, Michael Börsch

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69 Citations (Scopus)


A new technique combining scanning electrochemical microscopy (SECM) and single-molecule fluorescence spectroscopy was developed to accomplish locally and temporally defined pH adjustments in buffer solutions and on surfaces monitored by fluorescence alteration of pH-sensitive fluorophores in real time. Local pH gradients were created by electrochemical generation of H+ or OH-during redox reactions at ultramicro- or nanoelectrodes with radii from 5 μm to 35 nm. Ratiometric fluorescence measurements were performed with a confocal laser microscope using two detectors for different spectral regions. Time-resolved pH measurements were carried out with freely diffusing SNARF-1-dextran. For pH measurements on surfaces, total internal reflection fluorescence microscopy was used in combination with a CCD camera. The fluorophore SNAFL-succinimidyl ester was bound to amino-terminated octadecylsilane-coated coverslips. Local pH determinations could be accomplished with an accuracy of 0.2 unit. The measured pH profiles showed a strong dependence on the tip diameter, the buffer/mediator concentration ratio, and die tip-surface distance. As an application for bionanotechnology using SECM-induced pH changes on the molecular level, the proton-driven ATP synthesis by single membrane-bound F0F1-ATP synthases was investigated. ATP synthesis resulted in stepwise subunit rotation within the enzyme that was monitored by single-molecule fluorescence resonance energy transfer.

Original languageEnglish
Pages (from-to)3473-3481
Number of pages9
JournalAnalytical Chemistry
Issue number13
Publication statusPublished - 1 Jul 2004
Externally publishedYes

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