Real-Time pH Microscopy down to the Molecular Level by Combined Scanning Electrochemical Microscopy/Single-Molecule Fluorescence Spectroscopy
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- 作者：Frank-Mario Boldt ; Jü ; rgen Heinze ; Manuel Diez ; Jan Petersen ; and Michael Bö ; rsch
- 刊名：Analytical Chemistry
- 出版年：2004
- 出版时间：July 1, 2004
- 年：2004
- 卷：76
- 期：13
- 页码：3473 - 3481
- 全文大小：196K
- 年卷期：v.76,no.13(July 1, 2004)
- ISSN：1520-6882
文摘
A new technique combining scanning electrochemicalmicroscopy (SECM) and single-molecule fluorescencespectroscopy was developed to accomplish locally andtemporally defined pH adjustments in buffer solutions andon surfaces monitored by fluorescence alteration of pH-sensitive fluorophores in real time. Local pH gradientswere created by electrochemical generation of H+ or OH-during redox reactions at ultramicro- or nanoelectrodeswith radii from 5 
m to 35 nm. Ratiometric fluorescencemeasurements were performed with a confocal lasermicroscope using two detectors for different spectralregions. Time-resolved pH measurements were carriedout with freely diffusing SNARF-1-dextran. For pH measurements on surfaces, total internal reflection fluorescence microscopy was used in combination with a CCDcamera. The fluorophore SNAFL-succinimidyl ester wasbound to amino-terminated octadecylsilane-coated coverslips. Local pH determinations could be accomplishedwith an accuracy of 0.2 unit. The measured pH profilesshowed a strong dependence on the tip diameter, thebuffer/mediator concentration ratio, and the tip-surfacedistance. As an application for bionanotechnology usingSECM-induced pH changes on the molecular level, theproton-driven ATP synthesis by single membrane-boundF0F1-ATP synthases was investigated. ATP synthesisresulted in stepwise subunit rotation within the enzymethat was monitored by single-molecule fluorescence resonance energy transfer.
m to 35 nm. Ratiometric fluorescencemeasurements were performed with a confocal lasermicroscope using two detectors for different spectralregions. Time-resolved pH measurements were carriedout with freely diffusing SNARF-1-dextran. For pH measurements on surfaces, total internal reflection fluorescence microscopy was used in combination with a CCDcamera. The fluorophore SNAFL-succinimidyl ester wasbound to amino-terminated octadecylsilane-coated coverslips. Local pH determinations could be accomplishedwith an accuracy of 0.2 unit. The measured pH profilesshowed a strong dependence on the tip diameter, thebuffer/mediator concentration ratio, and the tip-surfacedistance. As an application for bionanotechnology usingSECM-induced pH changes on the molecular level, theproton-driven ATP synthesis by single membrane-boundF0F1-ATP synthases was investigated. ATP synthesisresulted in stepwise subunit rotation within the enzymethat was monitored by single-molecule fluorescence resonance energy transfer.